Methods for detecting shigella bacteria or antibodies to shigella bacteria with an immunoassay

ABSTRACT

This invention relates generally to in vitro methods for inducing or enhancing expression of enteric bacterial antigens and/or virulence factors thereby producing antigenically enhanced enteric bacteria, to methods for using antigenically enhanced enteric bacteria and to vaccines comprising antigenically enhanced enteric bacteria. A Shigella bacterium having enhanced antigenic property is disclosed. A culture medium useful for culturing the Shigella comprises 0.05% to 3% bile or 0.025% to 0.6% of one or more bile acids or salts. In addition a divalent cation chelator can also be included in the culture medium. The bacteria culture is in a growth phase at early log phase and between early log phase and stationary phase. The enhanced antigenic property is a higher level of an immunogenic antigen when compared to the antigenic property of the same bacteria grown on brain heart infusion broth. Also, antibodies to Shigella bacteria or Shigella bacteria in samples are detected by immunoassays.

This application is a divisional of application Ser. No. 08/866,586,filed May 30, 1997, now U.S. Pat. No. 5,858,352 which is a divisional ofapplication Ser. No. 08/538,543, filed Oct. 3, 1995, now U.S. Pat. No.5,681,736 which is a continuation in part of application Ser. No.08/318,409, filed Oct. 5, 1994, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to in vitro methods for inducing orenhancing expression of enteric bacterial antigens and/or virulencefactors thereby producing antigenically enhanced enteric bacteria, tomethods for using antigenically enhanced enteric bacteria and tovaccines comprising antigenically enhanced enteric bacteria.

BACKGROUND OF THE INVENTION

It is widely recognized that bacteria cultured in vitro usingconventional media and conditions express characteristics that aredifferent from the characteristics expressed during growth in theirnatural habitats, which includes in vivo growth of normal microflora orpathogens in an animal host. Therefore, such in vitro grown pathogenicbacteria might not be good for use as vaccine components. However, if itwere possible to define conditions that trigger or enhance expression ofvirulence factors, relevant physiology, or antigens includingouter-surface antigens then important products and therapeutics (e.g.,new antigens for vaccines, new targets for antibiotics, and novelbacterial characteristics for diagnostic applications) could be rapidlyidentified.

Several environmental factors have been identified which influenceexpression of virulence determinants in bacteria (Mekalanos, J. J., J.Bacteriol. 174:1-7, 1992). For instance, there is a long history ofresearch on the relationship between iron and virulence of bacteria, inparticular Shigella (Payne, Mol. MicroBiol., 3:1301-1306, 1989),Neisseria (Payne and Finkelstein, J. Clin. Microbiol., 6:293-297, 1977)and Pasteurella (Gilmour, et al., Vaccine, 9:137-140, 1991).

Other environmental signals that have been shown to control theexpression of coordinately regulated virulence determinants of a widevariety of bacteria in plants and animals include phenolic compounds,monosaccharide, amino acids, temperature, osmolarity, and other ions(Mekalanos, J. Bacteriol., 174:1-7, 1992).

Bacterial pathogens that enter an animal host through the intestine(i.e., oral route) encounter numerous host environment components andconditions that may affect bacterial physiology and expression ofvirulence factors. These components and conditions include bile, bileacids or salts, stomach pH, microaerophillic conditions (the intestinehas high CO₂, and low O₂), osmolarity and many others yet undefined.Invasive enteric pathogens require de novo protein synthesis toaccomplish internalization (Headley and Payne, Proc. Natl. Acad. Sci.,USA, 87:4179-4183, 1990). Therefore, bacteria may optimally producethese invasive factors only in response to certain environmental signalsnot ordinarily present in vitro. This hypothesis is supported by therecent report that antisera raised against conventionally grown C.jejuni had only a marginal effect on blocking in vitro internalization(Konkel, et al., J. Infect. Dis., 168:948-954, 1993). However,immunization of rabbits with extracts of Campylobacter grown in thepresence of epithelial cell monolayers, a condition enhancinginvasiveness, resulted in production of an antiserum that markedlyinhibited the internalization of the bacteria.

Researchers have been studying growth of bacteria in the intestinalenvironment to identify relevant virulence factors. For example,Campylobacter strain 81-176 grown in rabbit ileal loops expressesproteins not expressed under conventional laboratory in vitro cultureconditions (Panigrahi, et al., Infect. Immun., 60:4938-4944, 1992). Newor enhanced synthesis of proteins has been seen in Campylobactercultivated with INT 407 cell monolayers as compared to bacteria culturedin the absence of the epithelial cells (Konkel, et al., J. Infect. Dis.,168:948-954, 1993). Furthermore, these changes were temporallyassociated with increased invasiveness of C. jejuni. Other changes suchas cellular morphology, loss of flagella, expression of a new outermembrane protein and alteration in cell-surface carbohydrates wereinduced or enhanced in an avirulent strain of C. jejuni when passedintravenously and chorio-allantoically through chick embryos (Field, etal., J. Med. Microbiol., 38:293-300, 1993).

Other intestinal components, such as bile acids or salts, are known tobe inhibitory for some bacteria, but the bile acids may play anotherrole by affecting virulence expression by the bacterium.

Pope and Payne (93rd Am. Soc. Microbiol., B-147, 1993) reported thatShigella flexneri cultured in broth containing sodium chenodeoxycholatedemonstrated 3 to 5-fold enhanced infectivity of HeLa cell monolayers.They reported, however, that other bile salts and detergents includingcholate, glycocholate, taurodeoxycholate, the CHAPS series, digitoninand Triton X100 and sodium salts thereof, had no effect on theinvasiveness of S. flexneri. Moreover, their broth containingchenodeoxycholate also had no effect on the invasiveness of E. coli orother avirulent strains of Shigella.

Synthesis of new proteins by S. flexneri is also induced by altering pH,temperature and ionic composition of the growth medium (Mekalanos, J.Bacteriol., 174:1-7, 1992).

PCT application publication number WO 93/22423, published Nov. 11, 1993,discloses methods for growing bacteria on lipids, such asphosphatidylserine, or mucus and for the isolation of proteins whoseexpression is enhanced by growth in the presence of phosphatidylserine.This reference neither discloses nor suggests methods of the presentinvention for producing enteric bacteria having enhanced virulence orantigenic properties.

Vaccines against many enteric pathogens, such as Campylobacter andShigella, are not yet available but the epidemiology of these diseaseagents makes such vaccines an important goal. Shigellosis is endemicthroughout the world and in developing countries it accounts for about10 percent of the 5 million childhood deaths annually due to diarrhea.Campylobacter, although only recently identified as an enteric pathogenis now recognized as one of the major causes of diarrheal disease inboth the developed and underdeveloped countries. An estimated 400 to 500million Campylobacter diarrheas occur yearly, and over 2 million casesoccur in the United States.

Shigellosis is a consequence of bacterial invasion of the colonicmucosa. The invasion is associated with the presence of a plasmid foundin all invasive isolates (Sansonetti et al., Infect. Immun., 35:852-860,1982). A fragment of this plasmid contains the invasion plasmid antigen(Ipa) genes, Ipa A, -B, -C, and -D. Ipa B, -C, and -D proteins areessential for the entry process (Baudry et al., J. Gen. Microbiol.,133:3403-3413, 1987).

Ipa proteins are logical vaccine candidates although their protectiveefficacy has not been clearly established. Ipa B and Ipa C areimmunodominant proteins (Hale, et al., Infect. Immun., 50:620-629,1985). Furthermore, the 62 kDa Ipa B protein (the invasin that initiatescell entry and functions in the lysis of the membrane-bound phagocyticvacuole) (High, et al., EMBO J., 11:1991-1999, 1992) is highly conservedamong Shigella species. The prolonged illness observed in malnourishedchildren who have no significant mucosal antibody to Shigella Ipasuggests that the presence of mucosal antibody to Ipa may limit thespread and severity of infection.

Though a number of vaccine candidates for Shigella have been tested inanimals and humans, a successful one has not been found. In spite of thepotential significance of Ipa proteins in virulence, most vaccinecandidates developed against shigellosis are based on thelipopolysaccharide antigen, which carries the serotype-specificdeterminants. A parenterally administered polysaccharide-proteinconjugate vaccine has also been developed, but is yet to showsignificant protection in animals (Robbins et al., Rev. Inf. Dis.,13:S362-365, 1991). A similarly administered ribosomal vaccine doesinduce mucosal immunity, but its protective efficacy remains to bedemonstrated (Levenson et al., Arch. Allergy Appl. Immunol., 87:25-31,1988).

The pathogenesis of Campylobacter infections is not as well understoodas that of Shigella infections. Cell invasion studies in vitro (Konkel,et al., J. Infect. Dis., 168:948-954, 1993) and histopathologicexaminations (Russell, et al., J. Infect. Dis., 168:210-215, 1993)suggest that colonic invasion is also important. This conclusion isconsistent with the observation that diarrhea caused by Campylobactermay be severe and associated with blood in the stool. These activitiesmay be associated with the immunodominant 62 kDa flagellin protein. Arecent report indicates that the presence of flagella is essential forCampylobacter to cross polarized epithelial cell monolayers (Grant etal., Infect. Immun., 61:1764-1771, 1993).

No specific Campylobacter antigens have been established as protective.However, the low molecular weight (28-31 kDa) proteins, or PEB proteins,and the immunodominant flagellar protein are thought to hold promise inthis regard (Pavlovskis et al., Infect. Immun., 59:2259-2264, 1992;Blaser and Gotschilch, J. Bio. Chem., 265:14529-14535, 1990). Theimportance of the flagellar protein is indicated by its association withcolonization of the intestine and with the cross-strain protectionagainst infection within Lior subgroups (Pavlovskis et al., Infect.Immun., 59:2259-2264, 1992). However, a flagella protein basedCampylobacter vaccine may have to include the flagella protein antigenfrom the 8-10 most clinically relevant Lior serogroups.

Therefore, objects of the present invention include 1) in vitro cultureconditions for culturing or treating enteric bacteria which optimallyinduce or enhance invasive activities and/or certain cellularcharacteristics including cell surface characteristics; 2) correlatedaltered invasiveness or cellular characteristics including surfacecharacteristics with changes in antigenic profiles; 3) increasedvirulence of these organisms in small animal models; and 4) antiseraagainst organisms with enhanced invasiveness or altered characteristicsincluding surface characteristics that are more effective inneutralizing live organisms used for in vitro or in vivo challenges thanantisera prepared against conventionally grown bacteria. This inventionaddresses these needs and others.

None of the references discussed above teach or suggest the in vitromethods of the present invention nor the vaccines of the presentinvention comprising antigenically enhanced enteric bacteria. Citationor identification of any reference in this section or any other sectionof this application shall not be construed as indicative that suchreference is available as prior art to the invention.

SUMMARY OF THE INVENTION

This invention provides defined culture conditions and componentsincorporated into growth media of enteric bacteria to induce or enhancethe presence of virulence factors and other antigens. Preferably, suchantigens are immunogenic. More preferably, such immunogenic antigenscorrelate with indices of virulence.

Enteric bacteria are grown in the presence of conditions and componentssimulating certain in vivo conditions to which the organisms are exposedin nature. Methods of the present invention produce antigenicallyenhanced enteric bacteria with phenotypic changes such as increasedtotal protein per cell, new or increased individual proteins, altered orincreased surface carbohydrates, altered surface lipopolysaccharides,increased adhesive ability, increased invasive ability and/or increasedintracellular swarming. Moreover, methods of the present invention areadaptable to practical scale-up fermentations for commercial uses.

Said antigenically enhanced enteric bacteria can be used to produceprotective vaccines, such as inactivated whole cell or subunit vaccines,or for diagnostic purposes such as for the production of antibodies anddetection of pathogenic enteric bacteria or to produce antibiotics.Further, the antibodies induced by the enhanced enteric bacteria of thepresent invention may be used as passive vaccines.

Therefore, an object of the present invention is a method for producingenteric bacteria selected from the group consisting of Campylobactersp., Yersinia sp., Helicobacter sp., Gastrospirillum sp., Bacteroidessp., Klebsiella sp., Enterobacter sp., Salmonella sp., Shigella sp.,Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcus sp. andEscherichia coli, having enhanced antigenic properties comprising:growing enteric bacteria in vitro with a combination of conditionsincluding: a) 0.05% to 3% bile or 0.025% to 0.6% of one or more bileacids or salts thereof, at a temperature between 30° C. and 42° C.,until a growth phase at about early log phase, between early log andstationary phases, or at about stationary phase, in air ormicroaerophillic conditions, such as 5% to 20% CO₂ with 80% to 95% air,5% to 20% CO₂ with 80% to 95% N₂ ; or 5% to 10% O₂, 10% to 20% CO₂, with70% to 85% N₂ ; and optionally in the presence of a divalent cationchelator, such as, but not limited to 0 to 100 μM, preferably 25 μM, ofBAPTA/AM, 0 to 10 mM of EGTA, and 0 to 100 μM of EGTA/AM; or b) as in a)except in the presence of a divalent cation chelator, such as 1.0 to 100μM, preferably 25 μM, of BAPTA/AM, 0.5 to 10 mM of EGTA, or 1 to 100 μMof EGTA/AM, and without any bile, bile acids or bile salts.

According to the present invention, concentrations of any individualbile acid or salt thereof include 0.025% to 0.6%, preferably 0.05% to0.5%, more preferably 0.05% to 0.2%, most preferred is 0.05% or 0.1%.Preferred are methods wherein said bile acid or salt thereof comprisesdeoxycholate or glycocholate.

A further object of the invention is enteric bacteria selected from thegroup consisting of: Campylobacter sp., Yersinia sp., Helicobacter sp.,Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp.,Salmonella sp., Shigella sp., Aeromonas sp., Vibrio sp., Clostridiumsp., Enterococcus sp. and Escherichia coli, wherein said entericbacteria are grown in vitro under a combination of conditions to promoteenhanced antigenic properties, said conditions comprising: a) 0.05% to3% bile or 0.025% to 0.6% of one or more bile acids or salts thereof, ata temperature between 30° C. and 42° C., until a growth phase at aboutearly log phase, between early log and stationary phases, or at aboutstationary phase; in air or under microaerophillic conditions, such as5% to 20% CO₂ with 80% to 95% air, 5% to 20% CO₂ with 80% to 95% N₂, or5% to 10% O₂ with 10% to 20% CO₂, with 70% to 85% N₂ ; and optionally adivalent cation chelator, such as, but not limited to 0 to 100 μM,preferably 25 μM, of BAPTA/AM, 0 to 10 mM of EGTA, and 0 to 100 μM ofEGTA/AM, or b) as in a) except in the presence of a divalent cationchelator, such as 1.0 to 100 μM, preferably 25 μM, of BAPTA/AM, 0.5 to10 mM of EGTA, 1.0 to 100 μM of EGTA/AM, and without any bile, bileacids or bile salts.

Another object of the invention is a vaccine comprising a whole entericbacteria or components thereof, selected from the group consisting of:Campylobacter sp., Yersinia sp., Helicobacter sp., Gastrospirillum sp.,Bacteroides sp., Klebsiella sp., Enterobacter sp., Salmonella sp.,Shigella sp., Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcussp. and Escherichia coli, or an immunogenic fragment or derivativethereof, having enhanced antigenic properties; and optionally apharmaceutically acceptable carrier or diluent.

Preferred is the vaccine comprising whole, inactivated antigenicallyenhanced enteric bacteria. A further object of the invention is avaccine further comprising an adjuvant.

A further object of the present invention is directed to antibodies(including but not limited to antisera, purified IgG or IgA antibodies,Fab fragment, etc.) which are capable of specifically binding to atleast one antigenic determinant of an enteric bacteria of the presentinvention. Such polyclonal and monoclonal antibodies are useful asimmunoassay reagents for detecting enteric bacteria in an animal orbiological sample therefrom. The polyclonal and monoclonal antibodies ofthe present invention are also useful as passive vaccines for use inprotecting against enteric bacteria infections and diseases.

A further object of the invention is an in vitro method for assayingpotential antimicrobial agents comprising the steps of contactingenteric bacteria having enhanced antigenic properties selected from thegroup consisting of: Campylobacter sp., Yersinia sp., Helicobacter sp.,Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp.,Salmonella sp., Shigella sp., Aeromonas sp., Vibrio sp., Clostridiumsp., Enterococcus sp. and Escherichia coli, with said potential agentsand assaying the bacteriocidal or bacteriostatic effects.

Still a further object of the invention is an in vitro method fordetecting a host's production of antibodies or for the detection ofenteric bacteria in an animal or biological sample therefrom, comprisingthe steps of contacting a biological sample from a host with entericbacteria of the present invention having enhanced antigenic propertiesselected from the group consisting of: Campylobacter sp., Yersinia sp.,Helicobacter sp., Gastrospirillum sp., Bacteroides sp., Klebsiella sp.,Enterobacter sp., Salmonella sp., Shigella sp., Aeromonas sp., Vibriosp., Clostridium sp., Enterococcus sp. and Escherichia coli, antigensthereof or antibodies thereto and screening for antibody:antigeninteractions.

Another object of the present invention relates to a diagnostic kit fordetecting a host's production of antibodies to enteric bacteria or fordetecting enteric bacteria, comprising enteric bacteria having enhancedantigenic properties selected from the group consisting of:Campylobacter sp., Yersinia sp., Helicobacter sp., Gastrospirillum sp.,Bacteroides sp., Klebsiella sp., Enterobacter sp., Salmonella sp.,Shigella sp., Aeromonas sp., Vibrio sp., Clostridium sp., Enterococcussp. and Escherichia coli, or antibodies thereto and all other essentialkit components.

Preferred enteric bacteria that the various aspects of the presentinvention relate to are Campylobacter jejuni, Campylobacter coli,Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis,Escherichia coli, Shigella flexneri, Shigella sonnei, Shigelladysenteriae, Shigella boydii, Helicobacter pylori, Helicobacter felis,Gastrospirillum hominus, Vibrio cholerae, Vibrio parahaemolyticus,Vibrio vulnificus, Bacteroides fragilis, Clostridium difficile,Salmonella typhimurium, Salmonella typhi, Salmonella gallinarum,Salmonella pullorum, Salmonella choleraesuis, Salmonella enteritidis,Klebsiella pneumoniae, Enterobacter cloacae, and Enterococcus faecalis.Preferred Escherichia coli include but are not limited to eutero-toxic,entero-hemorrhagic, entero-invasive, entero-pathogenic or other strains.

The present invention is based, in part, on the surprising discoverythat antigenically enhanced enteric bacteria of the invention induceimmune responses that are cross-protective against a broader range ofstrains or serotypes of the same bacterial species than that induced bythe same enteric bacteria but grown using conventional culturingconditions. In at least one instance, the immune response induced by theantigenically enhanced enteric bacteria of the invention iscross-protective against a different species of enteric bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C graphically depict the results of high-performanceliquid chromatography of monosaccharides from surface extracthydrolysates of C. jejuni 81-176. FIG. 1A: Standards: Fucose "Fuc",N-acetyl-galactosamine "Gal", N-acetyl-glucosamine "GlcNac", galactose"Gal", glucose "Glc", Mannose "Man". FIG. 1B: surface extract ofconventionally grown bacteria "BHI". FIG. 1C: surface extracts ofbacteria grown according to methods of the present invention "DOC".

FIG. 2 pictorially depicts the results of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showing acomparison of proteins of whole cell (columns 1, 2 and 3) or surfaceextracts (col 5 and 6) of C. jejuni 81-176 conventionally grown "BHI" orgrown according to methods of the present invention (0.8% Oxgall bileacids "OX" or 0.1% deoxycholate "DOC").

FIG. 3 pictorially depicts the results of western blot analysis showinga comparison of proteins bound by ferret IgA-containing mucus producedby infection with whole cell C. jejuni 81-176. Whole cell C. jejuni81-176 conventionally grown, "1"; or whole cell C. jejuni 81-176 grownaccording to methods of the present invention: 0.8% Oxgall bile acids,"2", or 0.1% deoxycholate, "3"; or surface extracts of C. jejuni 81-176conventionally grown, "4"; or surface extracts of C. jejuni 81-176 grownaccording to methods of the present invention, 0.1% deoxycholate, "5".

FIG. 4 pictorially depicts the results of western blot analysis showinga comparison of proteins bound by flagellin-specific monoclonal antibody72c from whole cell C. jejuni 81-176 which were grown according tomethods of the present invention, "3"; conventionally grown, "2"; orgrown in a fermentor according to methods of the present invention, "1".

FIG. 5 pictorially depicts the results of SDS-PAGE showing a comparisonof lipopolysaccharides (LPS) of whole cell S. flexneri 2457Tconventionally grown, column "1", or grown according to methods of thepresent invention, 0.1% deoxycholate, column "2".

FIG. 6 graphically depicts the enhancement of immuno-cross reactivity ofC. jejuni grown according to methods of the present invention. C. jejuni81-176 cells grown conventionally or according to methods of theinvention as exemplified in Example 5 (DOC) were used to induceantibodies. The agglutination activity of the two types of antibodies(i.e., anti-C. jejuni 81-176 cultured in BHI and anti-C. jejuni 81-176cultured in DOC) against various C. jejuni serotypes are shown. SeeExample 32 in Section 9 for details.

FIGS. 7A, 7B and 7C graphically depict the efficacy of a vaccinecomprising inactivated C. jejuni 81-176 whole cells in protecting miceagainst a nasally delivered challenge of live C. jejuni 81-176 cells.Mice were vaccinated with phosphate buffered saline (PBS; solid line),PBS plus LT adjuvant (Adjuvant; dash line), formalin-inactivated C.jejuni 81-176 whole cells that were grown and harvested according toExample 5 without adjuvant (CWC; open circle/solid line) or with LTadjuvant (CWC+Adjuvant; solid circle/solid line). The vaccine efficacieswas examined using the intestinal colonization assay. FIG. 7A (toppanel) shows the results of the protection afforded by the vaccinationsusing three oral doses of 10⁵ inactivated bacterial particles per dose.FIG. 7B (middle panel) shows the results of the protection afforded bythe vaccinations using three oral doses of 10⁷ inactivated bacterialparticles per dose. FIG. 7C (bottom panel) shows the results of theprotection afforded by the vaccinations using three oral doses of 10⁹inactivated bacterial particles per dose. See Example 34 in Section 11for details.

FIGS. 8A, 8B and 8C graphically depict the efficacy of vaccinecomprising inactivated C. jejuni 81-176 whole cells in protecting miceagainst an orally delivered challenge of live C. jejuni 81-176 cells.Mice were vaccinated as described in the brief description of FIGS. 7A,7B and 7C. The vaccine efficacy was examined using the intestinalcolonization assay.

FIG. 8A (top panel) shows the results of the protection afforded by thevaccinations using three oral doses of 10⁵ inactivated bacterialparticles per dose. FIG. 8B (middle panel) shows the results of theprotection afforded by the vaccinations using three oral doses of 10⁷inactivated bacterial particles per dose. FIG. 8C (bottom panel) showsthe results of the protection afforded by the vaccinations using threeoral doses of 10⁹ inactivated bacterial particles per dose. See Example34 in Section 9 for experimental details.

FIG. 9 graphically depicts the effect of the growth phase of theShigella flexneri culture on the invasiveness of the Shigella flexneri2457T cells. Shigella flexneri 2457T cells were grown conventionally(BHI), or according to the methods of the present invention asexemplified by Example 9 (DOC-EL)--that is harvesting the cells when theCulture is in early log phase--or according to Example 9 but allowingthe culture to reach late log phase before harvesting the cells(DOC-LL). The invasiveness of these different preparations of cells areshown. See Example 35 in Section 12 for details.

FIG. 10 graphically depicts the enhancement: of invasiveness of Shigellacells when they are cultured using the methods of the present invention.S. sonnei and S. dysenteriae 3818 were cultured conventionally (BHI) oraccording to the methods of the present invention as exemplified inExample 9. The invasiveness of these different preparations of Shigellacells against INT-407 cells are shown. See Example 35 in Section 12 fordetails.

FIG. 11 graphically depicts the enhancement: of immuno-cross reactivityof Shigella grown according to methods of the present invention. S.flexneri 2457T grown according to methods of the present invention asexemplified in Example 9 was used to induce antibodies. Theagglutination activity of the induced antibodies against S. flexneri, S.sonnei, S. dysenteriae and S. boydii grown conventionally (BHI) oraccording to the methods of the present invention as exemplified inExample 9 serotypes are shown. See Example 36 in Section 12 for details.

FIG. 12 graphically depicts the effect of bile concentration and thegrowth phase of the Helicobacter pylori culture on the adhesiveness ofHelicobacter pylori. NB3-2 cells. H. pylori NB3-2 cells were grown inculture medium containing 0%, 0.025%, 0.05% or 0.1% bile and harvestedat 8, 10, 12 and 18 h after inoculation. The invasiveness of thesedifferent preparations of H. pylori NB3-2 cells against INT-407 cellsare shown. See Example 38 in Section 14 for details.

FIG. 13 graphically depicts the effect of bile concentration and thegrowth phase of the Helicobacter pylori culture on the adhesiveness ofHelicobacter pylori G1-4 cells. H. pylori G1-4 cells were grown inculture medium containing 5 0%, 0.1% or 0.2% bile and harvested at 6, 8,10, :.2, 14 and 16 h after inoculation. The invasiveness of thesedifferent preparations of H. pylori G1-4 cells against INT-407 cells areshown. See Example 38 in Section 15 for details.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention relate to growing enteric bacteriain vitro in the presence of a combination of certain conditions withcertain components selected to induce or enhance the expression ofantigens and/or virulence factors.

As used herein and in the claims the term "enteric" refers to bacterianormally found in or associated with any part of an animal'sgastrointestinal tract and any bacteria that causes an infection in anypart of an animal's gastrointestinal tract. Such enteric bacteriainclude both gram positive and gram negative bacteria.

The terms "components" and "conditions" as used herein and in the claimsrelate to many factors associated with an enteric bacterium's natural invivo environment and other factors. Such components and conditionsinclude, but are not limited to, bile, bile acids or salts thereof ortheir biological precursors such as cholesterol, pH, microaerophilliccondition, osmolarity, and harvesting or collecting the bacteria at adesired bacterial growth phase.

The term "antigens" and its related term "antigenic" as used herein andin the claims includes antigens or antigenic characteristics including,but not limited to, macromolecules contributing to cellular morphologyor cell motility; proteins; more particularly surface proteins,lipopolysaccharides and carbohydrates. Preferably said antigens areimmunogenic.

The term "immunogenic" as used herein and in the claims refers to theability to induce antibody production in an animal after said animal isexposed to a composition comprising whole bacteria produced by thepresent invention or a fragment of said whole bacterium.

The term "antigenically enhanced" or "enhanced antigenic properties" or"enhanced" as used herein and in the claims refers to the antigenicstate of enteric bacteria grown according to the methods of the presentinvention. Such bacteria have higher levels of certain immunogenicantigens and/or new immunogenic antigens as compared to the samebacteria grown using conventional methods.

The term "conventional" as used herein and in the claims relates to whatis known in the prior art.

The term "microaerophillic conditions" as used herein and in the claimsrefers to anaerobic conditions or elevated CO₂ levels, such as 5% to 20%CO₂ with 80% to 95% air; 5% to 20% CO₂ with 80% to 95% N₂ ; or 5% to 10%O₂ with 10% to 20% CO₂ with 70% to 85% N₂.

The term "virulence" as used herein and in the: claims refers to thosefactors of an enteric bacteria associated with the ability to adhere toand/or to invade and/or to survive in a host and/or cause a pathologicalcondition.

The term "immuno-cross protective" as used herein and in the claimsrefers to the ability of the immune response induced by one bacterialstrain or serotype, whole cell or otherwise, to prevent or attenuateinfection of the same host by a different bacterial strain, serotype, orspecies of the same genus.

The term "immuno-cross reactive" as used herein and in the claims refersto the ability of the humoral immune response (i.e., antibodies) inducedby one bacterial strain or serotype, whole cell or otherwise, to crossreact with (i.e., the antibody binding) a different bacterial strain,serotype, or species of the same genus. Immuno-cross reactivity isindicative of the bacterial immunogen's potential for immuno-crossprotection and vice versa.

The term "host" as used herein and in the claims refers to either invivo in an animal or in vitro in animal cell cultures. The term "animal"as used herein and in the claims includes but is not limited to allwarm-blooded creatures such as mammals and birds (e.g., chicken, turkey,duck etc.)

According to the invention, in a vaccine comprising antigenicallyenhanced enteric bacteria or an immunogenic fragment or derivativethereof, the enteric bacteria may be either live bacteria or may beinactivated and may further comprise an adjuvant, such as, but notlimited to, alum, oil-water emulsion, heat labile toxin fromenterotoxigenic E. coli (LT) nontoxigenic forms thereof (eg. mLT) and/orindividual subunits thereof, Bacille Calmette-Guerin (BCG), or Fruend'sadjuvant and may also further comprise a suitable pharmaceuticalcarrier, including but not limited to saline, dextrose or other aqueoussolution. Other suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences, Mack Publishing Company, a standardreference text in this field. As used herein and in the claims, the term"vaccine" also encompasses "passive vaccines," which comprise antibodiesthat specifically bind pathogens against whose infections or diseasesprojection is sought.

The term "inactivated bacteria," as used herein and in the claim, refersto enteric bacteria that are incapable of infection and/or colonizationand encompasses attenuated as well as killed bacteria. Attenuatedbacteria may replicate but cannot cause infection or disease.Inactivation of said bacteria may be accomplished by any methods knownby those skilled in the art. For example, the bacteria may be chemicallyinactivated, such as by formalin fixation, or physically inactivatedsuch as by heat, sonication or irradiation, so that they are renderedincapable of replication and/or infection and/or causing disease.

An effective amount of the vaccine should be administered, in which"effective amount" is defined as an amount of enteric bacteria or animmunogenic fragment or derivative thereof that is capable of producingan immune response in a subject. The amount needed will vary dependingupon the antigenicity of the bacteria, fragment, or derivative used, andthe species and weight of the subject to be vaccinated, but may beascertained using standard techniques. In preferred, non-limitingembodiments of the invention, an effective amount of vaccine produces anelevation of anti-bacterial antibody titer to at least two times theantibody titer prior to vaccination. In a preferred, specific,non-limiting embodiment of the invention, approximately 10⁷ to 10¹¹bacteria and preferably 10⁸ to 10¹⁰ bacteria are administered to a host.Preferred are vaccines comprising inactivated whole bacteria.

The term "effective amount" as applied to passive vaccines is an amountof antibody that is capable of preventing or attenuating a bacterialdisease or infection. The amount needed will vary depending upon thetype of antibody and the antibody titer, and the species and weight ofthe subject to be vaccinated, but may be ascertained using standardtechniques.

Vaccines of the present invention may be administered locally and/orsystemically by any method known in the art, including, but not limitedto, intravenous, subcutaneous, intramuscular, intravaginal,intraperitoneal, intranasal, oral or other mucosal routes.

Vaccines may be administered in a suitable, nontoxic pharmaceuticalcarrier, may be comprised in microcapsules, and/or may be comprised in asustained release implant.

Vaccines may desirably be administered at several intervals in order tosustain antibody levels.

Vaccines of the invention may be used in conjunction with otherbacteriocidal or bacteriostatic methods.

Antibodies of the invention may be obtained by any conventional methodsknown to those skilled in the art, such as but not limited to themethods described in Antibodies A Laboratory Manual (E. Harlow, D. Lane,Cold Spring Harbor Laboratory Press, 1989). In general, an animal (awide range of vertebrate species can be used, the most common beingmice, rats, guinea pig, hamsters and rabbits) is immunized with a wholecell or immunogenic fragment or derivative of an antigenically enhancedenteric bacteria of the present invention in the absence or presence ofan adjuvant or any agent that would enhance the immunogen'seffectiveness and boosted at regular intervals. The animal serum isassayed for the presence of desired antibody by any convenient method.The serum or blood of said animal can be used as the source ofpolyclonal antibodies.

For monoclonal antibodies, animals are treated as described above. Whenan acceptable antibody titre is detected, the animal is euthanized andthe spleen is aseptically removed for fusion. The spleen cells are mixedwith a specifically selected immortal myeloma cell line, and the mixtureis then exposed to an agent, typically polyethylene glycol or the like,which promotes the fusion of cells. Under these circumstances fusiontakes place in a random selection and a fused cell mixture together withunfused cells of each type is the resulting product. The myeloma celllines that are used for fusion are specifically chosen such that, by theuse of selection media, such as HAT: hypoxanthine, aminopterin, andthymidine, the only cells to persist in culture from the fusion mixtureare those that are hybrids between cells derived from the immunizeddonor and the myeloma cells. After fusion, the cells are diluted andcultured in the selective media. The culture media is screened for thepresence of antibody having desired specificity towards the chosenantigen. Those cultures containing the antibody of choice are cloned bylimiting dilution until it can be adduced that the cell culture issingle cell in origin. The antibodies of the present invention have useas passive vaccines against enteric bacteria infections and diseases.

Methods for the detection of antibodies or said bacteria in a hostinclude immunoassays. Such immunoassays are known in the art andinclude, but are not limited to radioimmunoassays (RIA), enzyme-linkedimmunosorbent assays (ELISA), fluorescent immunoassays, and fluorescencepolarization immunoassays (FPIA).

Another embodiment includes diagnostic kits comprising all of theessential reagents required to perform a desired immunoassay accordingto the present invention. The diagnostic kit may be presented in acommercially packaged form as a combination of one or more containersholding the necessary reagents. Such a kit comprises an enteric bacteriaof the present invention, and/or a monoclonal or polyclonal antibody ofthe present invention in combination with several conventional kitcomponents. Conventional kit components will be readily apparent tothose skilled in the art and are disclosed in numerous publications,including Antibodies A Laboratory Manual (E. Harlow, D. Lane, ColdSpring Harbor Laboratory Press, 1989). Conventional kit components mayinclude such items as, for example, microtiter plates, buffers tomaintain the pH of the assay mixture (such as, but not limited to Tris,HEPES, etc.), conjugated second antibodies, such as peroxidaseconjugated anti-mouse IgG (or any anti-IgG to the animal from which thefirst antibody was derived) and the like, and other standard reagents.

Methods of the present invention include growing enteric bacteria in asuitable basal essential culture medium, such as but not limited tocommercially available brain heart infusion broth "BHI", Luria broth"LB", sheep blood agar "SBA", Brucella broth, Meuller-Hinton broth,proteose peptone beef extract broth, etc., with various conditions andcomponents including but not limited to 0.05% to 3% bile or 0.025% to0.6% of one or more bile acids or salts thereof or biological precursorsthereof such as cholesterol, at a temperature between 30° C. and 42° C.,until a growth phase at about early log phase, between early log andstationary phases, or at about stationary phase, in air or undermicroaerophillic conditions, such as 5% to 20% CO₂ with 80% to 95% air;5% to 20% CO₂ with 80% to 95% N₂ ; or 5% to 10% O₂ with 10% to 20% CO₂with 70% to 85% N₂ ; and optionally in the presence of a divalent cationchelator, such as, but not limited to 0 to 100 μM, preferably 25 μM, ofBAPTA/AM (2'(ethylenedioxy)dianiline n,n,n',n'-tetraaceticacid/acetoxymethyl ester; Molecular Probes, Eugene, OR), 0 to 10 mM ofEGTA (ethylenebis(oxyethylenenitrilo)-tetraacetic acid; Sigma ChemicalCo., St. Louis, Mo.), 0 to 100 μM of EGTA/AM(ethylenebis(oxyethylenenitrilo)-tetraacetic acid/acetoxymethyl ester;Molecular Probes, Eugene, OR); the resulting combination of conditionsand components producing antigenically enhanced enteric bacteria.

According to another embodiment, the methods of the present inventionalso include growing enteric bacteria as described immediately aboveexcept in the presence of a divalent cation chelator, such as, but notlimited to 1.0 to 100 μM, preferrably 25 μM, of BAPTA/AM, 0.5 to 10 mMof EGTA, or 1.0 to 100 μM of EGTA/AM; but without any bile, bile acidsor bile salts.

Bile or bile acids or salts thereof useful for the present inventioninclude any natural bile compound secreted by the liver and normallyconcentrated in the gall bladder as well as synthetic bile acids knownby those skilled in the art, such as but not limited to "OXGALL" (DifcoLaboratories, Detroit, Mich.), bovine bile (Sigma Chemicals, St. Louis,Mo.) or other commercially available preparations, cholic, deoxycholic,taurocholic and glycocholic acids. Preferred is deoxycholate (DOC), acommercially available bile acid present in vivo in the distal smallintestine and large intestine sites colonized by some enteric bacteria.Also preferred is glycocholate (GC).

According to the present invention, enteric bacterial cultures selectedfrom the group of Campylobacter sp., Yersinia sp., Helicobacter sp.,Gastrospirillum sp., Bacteroides sp., Klebsiella sp., Enterobacter sp.,Salmonella sp., Aeromonas sp., Vibrio sp., Shigella sp., Clostridiumsp., Enterococcus sp., and Escherichia coli can be prepared as frozenstocks by methods generally known to those skilled in the art andmaintained at -80° C. for future use. For instance, stocks ofCampylobacter jejuni can be prepared by growing the organism ontrypticase soy agar containing 5% defribinated sheep erythrocytes (SBA),at 37° C. in 5% 10% CO₂ 85% N₂ (microaerophilic condition, "MC") for 20h. Stocks of Escherichia coli, Salmonella typhimurium, Helicobacterpylori and Shigella flexneri can be prepared by growing the organism inbrain heart infusion broth ("BHI"). Bacteria can be harvested forfreezing by any known method, for instance by swabbing the culture andresuspending in BHI containing 30% glycerol. Cultures for analyticalexperiments or for production fermentations can be prepared by anygenerally known methods, such as by growing the organism on BHI with1.5% agar at 37° C. under MC or atmospheric conditions and thentransferring a single colony to broth and culturing according to methodsof the present invention described herein. Bacteria can be harvested foruse by any method generally known to those skilled in the art, such asby centrifugation.

In preferred embodiments, antigenically enhanced cells of Campylobactersp., preferably of the species jejuni or coli and most preferably of thejejuni strain 81-176, are grown in a basal essential culture medium,preferably BHI broth, additionally comprising about 0.1% DOC or about0.8% bile at 37° C. in a mixture of about 10 to 20% CO₂ with about 80 to90% air and harvested after the growth of the culture has reached aboutlate log phase to about stationary phase, typically about 20 h afterinoculation.

In other preferred embodiments, antigenically enhanced cells of Shigellasp., preferably of the species flexneri or dysentariae and mostpreferably of the flexneri strain 2457T, are grown in a basal essentialculture medium, preferably BHI broth, additionally comprising about 0.1%DOC or about 0.8% bile at 37° C. in air and harvested after the growthof the culture has reached about early log phase, typically about 30 minafter inoculation with a culture that is at early to mid log phase.

In further preferred embodiments, antigenically enhanced cells ofHelicobacter pylori, preferably of the strain ATCC 49503, NB3-2 or G1-4,are grown in a basal essential culture medium, preferably BHI broth,additionally comprising about 0.05% to about 0.2% bile or about 0.05%glycocholate (GC) at 37° C. in a mixture of about 5% to 20% CO₂ withabout 80% to 95% air, or about 10% CO₂ with about 5% O₂ with about 85%N₂ and harvested after the growth of the culture has reached about logor about stationary phase. In a more preferred embodiment, the cells areharvested after the culture has reached about log phase.

Enteric bacteria cultured according to the methods of the presentinvention have altered morphologies, and/or cell motilities and/orproduce certain new proteins, lipopolysaccharides and/or carbohydratesand/or such macromolecules at altered levels compared to cells culturedin basal medium alone. Optimum cultural conditions that enhance cellyield and the indices of pathogenicity can be identified. Utilizingthese cultural conditions, virulence-associated antigens that areenhanced or induced can be identified.

Motility and gross morphological changes can be seen by microscopicexamination of either untreated or stained bacteria. It is possible thatother morphological changes might result from methods of the presentinvention as could be seen through electron microscopy or fluorescencemicroscopy.

The morphology and mucus-like characteristics of the enteric bacteriacultured according to methods of the present invention suggest thatcapsule and/or surface layer expression might be induced. To test forcapsule production, phenol extracts of surface components, such asproteins, carbohydrates and lipopolysaccharides, can be prepared. Theenhanced carbohydrates can be seen by high pressure liquidchromatography (HPLC).

Protein profiles of outer membranes prepared from enteric bacteria grownunder virulence enhancing growth condictions of the present inventioncan be characterized by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and compared to those from organisms grown inconventional media. SDS-PAGE is conducted to evaluate changes induced inbacterial cellular and extracted proteins in response to antigenenhancing or altering conditions. These data offer qualitative andquantitative information concerning surface changes associated withincreased invasiveness or altered antigenicity.

The immunogenic potential of induced or altered protein antigens can beidentified by Western Blotting. Immunogenicity of induced or alteredbacterial proteins identified by SDS-PAGE can be evaluated by thegenerally accepted techniques of Western Blotting as described below.Any source of antibody can be used, such as convalescent immune rabbitor ferret sera (source of antibody from animals infected orally withlive organisms grown conventionally or according to methods of thepresent invention), intestinal mucus (source of IgA antibody),polyclonal antisera, or a monoclonal antibody, for instance one that iscross-reactive with C. jejuni flagellin, for assaying bacterialantigens.

The increased production of several of these bacterial antigens isaccompanied with enhancement of properties associated with virulence.These properties include adhesion to and invasion of cultured humanintestinal epithelial cells, and Congo red dye binding. The Congo reddye binding assay is a generally accepted method predictive of virulenceand is described below and in Andrews et al., (Infect. Immun.,60:3287-3295, 1992); and Yoder (Avian Dis., 33:502-505, 1989). Bacterialbinding of Congo red indicates ability to bind hemin, this ability tobind hemin is correlated with virulence. Congo red binding alsocorrelates with enhanced bacterial invasion of epithelial cells.

Previously it has been shown that most conventionally grown C. jejuniLior serotypes are not immuno-cross reactive. Based on this information,it would be necessary to prepare and administer a number of vaccines ofseveral Lior serotype strains or a combination vaccine to obtaingeneralized protection against Campylobacter. Antigenically enhancedCampylobacter produced according to methods of the present invention areimmuno-cross reactive against a broader range of Lior serotypes thanthat obtained using conventionally grown Campylobacter. Similarly,antigenically enhanced Shigella produced according to the methods of thepresent invention are immuno-cross reactive and immuno-cross protectiveagainst a broader range of Shigella species than that obtained usingconventionally grown Shigella. These immuno-cross reactive andimmuno-cross protective properties are indicated by the results ofagglutination assays and vaccine studies described below.

Methods of the present invention for production of antigenicallyenhanced enteric bacteria correlate to enhanced virulence in smallanimal models. Several domestic animals can be used as models ofCampylobacter disease in humans. The most studied from the standpoint ofimmunization efficacy is the reversible intestinal tie, adult rabbitdiarrhea (RITARD Model) of Caldwell, et al., (Infect. Immun.,42:1176-1182, 1983). This model has also demonstrated the association ofLior serotypes with cross-strain protection. However, this modelmeasures resistance to colonization rather than resistance to disease.The ferret model of Bell et al. (Infect. Immun., 58:1848-1852, 1990) maybe a more useful model because this animal does develop disease symptomslike those of Campylobacter disease in humans. A more recent model isthe mouse colonization model of Bagar (Infection and Immunity,63:3731-3735, 1995). This model involves immunization followed by nasalor oral challenges with live Campylobacter to the immunized mouse andthen examination of intestinal colonization by monitoring for thepresence of Campylobacter in fecal matter. With Shigella, the mousenasal challenge assay as described by Mallett et al. (see infra Section13) is used to assess virulence and vaccine efficacy. With Helicobacterpylori, the Helicobacter felis gastric colonization model described byChen et al. (Lancet, 339:1120-1121, 1992) is used to evaluate thevaccine potential of H. pylori produced by the methods of the presentinvention.

There are other animal models relevant to enteric bacterial invasion andinfection and they can be used to test for vaccine efficacy of theenhanced enteric bacteria produced according to the methods of thepresent invention.

The antigenically enhanced enteric bacteria produced by the methods ofthe present invention are used to prepare prototype killed whole-cell orsubunit vaccines. These vaccines when administered to animals can beshown to induce antibodies and thus establish the vaccines' protectivepotential. Elevation or induction of these antigens in a bacterial cellproduces cells that make more efficacious vaccines.

Vaccine candidate preparations produced by the methods of the presentinvention can be used with various mucosal immunization strategies toinduce an intestinal immune response. Successful immunization protocolscan then be used to protect animals challenged with the pathogens withor without enhanced antigenicities. Also vaccines can be formulated andtested as combined vaccines, for example Shigella and Campylobactercells or components thereof can be combined as a single vaccine.

The experimental approaches described below enable the detection ofchanges in surface antigens and other characteristics which areassociated with enhanced invasiveness. These changes are bothqualitative and quantitative. Most importantly, these experimentsestablish that the methods of the present invention enhance invasivenessand induce immunogenically relevant antigens. Such antigenicallyenhanced bacteria induce an immunogenic response which is protectiveagainst infections by a broader range of bacterial strains, serotypesand/or species and are therefore useful as efficacious vaccines ascompared to conventionally grown bacteria.

Several well-established models generally known to those skilled in theart exist and are useful to evaluate enhanced antigenic properties,virulence of bacteria and vaccine efficacy as described below.

Non-limiting examples of the present invention are described below.

EXAMPLES: METHODS FOR PRODUCING ENHANCED ANTIGENIC BACTERIA Example 1

Campylobacter jejuni strain 81-176 was streaked on blood agar plates(containing trypticase soy agar, plus 5% defibrinated sheeperythrocytes) and incubated in a microaerophilic GasPak jar (BBL,Cockeysville, Md.) for 20 h at 37° C. Lawns of bacteria were removed byswabbing and inoculated into flasks containing 1 liter of BHI mediumpre-equilibrated to 10% CO₂, 90% air, with 0.8% OXGALL (Difco, Detroit,Mich.). Cultures were incubated for 20 h with shaking at 37° C. in aclosed flask at 10% CO₂, 90% air and then harvested as described above.

Example 2

Campylobacter jejuni strain 81-176 was streaked on blood agar plates(containing trypticase soy agar, plus 5% defibrinated sheeperythrocytes) and incubated in a microaerophilic GasPak jar (BBL,Cockeysville, Md.) for 20 h at 37° C. Lawns of bacteria were removed byswabbing and inoculated into flasks containing 1 liter of BHI mediumpre-equilibrated to 10% CO₂ with 0.01% to 0.1% sodium deoxycholate(DOC). (It is critical to prepare and autoclave the DOC as a stocksolution separate from the BHI medium, and to aseptically add it to theBHI medium to a final concentration of 0.01% to 0.1% immediately beforeinoculation of the bacteria). Cultures were incubated for various timesup to 20 h with shaking at 37° C. in 5% O₂, 10% CO₂, 85% N₂, thenharvested as described above.

Example 3

Campylobacter jejuni strain 81-176 was streaked on blood agar plates(containing trypticase soy agar, plus 5% defibrinated sheeperythrocytes) and incubated in a microaerophilic GasPak jar (BBL,Cockeysville, Md.) for 20 h at 37° C. Lawns of bacteria were removed byswabbing and inoculated into flasks containing 1 liter of Brucella brothpre-equilibrated to 10% CO₂ with 0.01% to 0.1% sodium deoxycholate.Cultures were incubated for 20 h with shaking at 37° C. in 5% O₂, 10%CO₂, 85% N₂ and then harvested as described above.

Example 4

Campylobacter jejuni strain 81-176 was streaked on blood agar plates(containing trypticase soy agar, plus 5% defibrinated sheeperythrocytes) and incubated in a microaerophilic GasPak jar (BBL,Cockeysville, Md.) for 20 h at 37° C. Lawns of bacteria were removed byswabbing and inoculated into flasks containing 1 liter of Mueller-Hintonbroth pre-equilibrated to 10% CO₂, with 0.01% to 0.1% sodiumdeoxycholate. Cultures were incubated for 20 h with shaking at 37° C. in5% O₂, 10% CO₂, 85% N₂ and then harvested as described above.

Example 5

Campylobacter jejuni strain 81-176 was streaked on blood agar plates(containing trypticase soy agar, plus 5% defibrinated sheeperythrocytes) and incubated in a microaerophilic GasPak jar (BBL,Cockeysville, Md.) for 20 h at 37° C. Lawns of bacteria were removed byswabbing and inoculated into flasks containing 1 liter of BHI mediumpre-equilibrated to 10% CO₂, with 0.1% sodium deoxycholate. Cultureswere incubated for 20 h with slow stirring at 37° C. in 10% CO₂, 90% airand then harvested as described above.

Example 6

Vibrio cholerae is streaked on BHI agar plates (containing trypticasesoy agar, plus 5% defibrinated sheep erythrocytes) and incubated in airfor 20 h at 37° C. Lawns of bacteria are removed by swabbing andinoculated into flasks containing 1 liter of BHI medium with 0.1% sodiumdeoxycholate. Cultures are incubated for 20 h with shaking at 37° C. inair, then harvested as described above.

Example 7

Salmonella cholerasius is streaked on BHI agar plates and incubated inair for 20 h at 37° C. Lawns of bacteria are removed by swabbing andinoculated into flasks containing 1 liter of BHI medium with 0.1% sodiumdeoxycholate. Cultures are incubated for 20 h with shaking at 37° C. inair, then harvested as described above.

Example 8

Salmonella typhimurium is streaked on Luria broth agar plates andincubated for 20 h at 37° C. in air. Lawns of bacteria are inoculatedinto flasks containing 1 liter of BHI medium with 0.1% sodiumdeoxycholate. Cultures are incubated for 20 h with shaking at 37° C. in10% CO₂, 90% air. One colony is transferred into 1 liter of LBcontaining 0.1% DOC and incubated in a closed top flask at 37° C. withslow shaking. After 12 h, 60 ml of the culture is diluted into 1 literof the same fresh prewarmed medium and incubated a further 30 min, andthen is harvested as described above.

Example 9

Shigella flexneri 2457T was streaked on Congo red agar plates andincubated for 20 h at 37° C. in air. One red colony was inoculated intoflasks containing 1 liter of BHI media and incubated with shaking for 12h. 50 ml of this culture was then used to inoculate 250 ml of prewarmedBHI containing 0.1% sodium deoxycholate. The culture was incubated withshaking for 4 h at 37° C. in air. This culture was then diluted to anOD₆₀₀ of about 0.17 with prewarmed BHI containing 0.1% DOC and incubatedfor 30 min with shaking at 37° C. in air, and then harvested asdescribed above.

Example 10

Campylobacter jejuni 81-176, 81-116, or HC (in BHI with 30% glycerol)was rapidly thawed and plated on sheep blood agar (SBA, 0.1 ml perplate). The inoculated plates were incubated in a GasPak jar with aCampyPak Plus microaerophilic environment generator (BBL, Cockeysville,Md.) for 20 h at 37° C. The bacterial lawn was removed from the platesby swabbing, and the bacteria were resuspended in 10 ml of BHI. Thebacterial suspension was inoculated into 1 liter of BHI broth alone orBHI broth containing 0.1% DOC, pre-equilibrated to 10% CO₂, 90% air, ina 2 liter flask. The inoculum was added to preequilibrated medium untilthe OD₆₂₅ is equal to 0.05. The inoculated flask was returned to the 10%CO₂ with 90% air and stirred slowly for 20 h at 37° C. At this point thebacteria were harvested as described above.

Example 11

Helicobacter pylori was added to BHI broth plus 4% bovine calf serum.After inoculation the flasks were flushed with 5% O₂, 10% CO₂, 85% N₂and incubated for 22 h at 37° C. with shaking. After this incubation,2.5 ml of the culture was transferred to a flask containing BHI brothwith 4% bovine calf serum or the same medium additionally containing0.05% sodium glycocholate. These cultures were again flushed with themicroaerophilic gas mixture (5% 0₂, 10% CO₂ 85% N₂), and incubated 20-24h at 37° C. The cells were harvested as described above.

Example 12

Salmonella typhimurium (in LB with 30% glycerol) was streaked on a LBagar plate and cultured for 18-20 h at 37° C. in air. One colony waspicked and transferred into 1 liter of LB or LB containing 0.1% DOC inflasks that are flushed with 10% CO₂, 5% CO₂, 85% N₂, sealed andincubated for 12 h at 37° C. with shaking. The bacteria were thendiluted in the same media to OD₆₀₀ of 0.17 and incubated under identicalconditions until the culture reaches early log phase, typically 30 minafter the dilution. Cells were harvested as described above.

Example 13

Salmonella typhimurium is streaked on a LB agar plate and cultured for18-20 h at 37° C. in air. One colony is picked and transferred into 1liter of LB or LB containing 0.1% DOC and incubated for 12 h at 37° C.in air. The culture is then diluted (1/5) in the same fresh media andincubated a further 4 hours under identical conditions. The cultures arethen diluted in the same fresh media to OD₆₀₀ of 0.17 and incubatedunder identical conditions until the culture reaches log phase,typically 30 minutes after the dilution. Cells are harvested asdescribed above.

Example 14

Klebsiella pneumoniae is streaked on a BHI agar plate and incubated18-20 h at 37° C. in air. One colony is picked and inoculated into 1liter of BHI or BHI containing 0.1% DOC and shaken for 12 h at 37° C. inair. The bacteria are then diluted in the same media to OD₆₀₀ of 0.17and grown for 30 min further and then harvested as described above.

Example 15

Enterobacter cloacae is streaked on a BHI agar plate and incubated at37° C. in air for 18-20 h. One colony is inoculated into 1 liter of BHIor BHI containing 0.1% DOC and shaken for 12 h at 37° C. The bacteriaare then diluted in the same media to OD₆₀₀ of 0.17 and grown for 30 minfurther and then harvested as described above.

Example 16

Escherichia coli strain 0157:H7 was streaked on sheep blood agar plateand incubated at 37° C. in air for 18-20 h. One colony was inoculatedinto 1 liter BHI or BHI containing 0.1% to 0.2% DOC flask and shaken for12 h at 37° C. The bacteria were then diluted to OD₆₀₀ of 0.17 and grownfor 30 min further and then harvested as described above.

Example 17

Enterococcus faecalis is streaked on sheep blood agar plate andincubated at 37° C. in air for 18-20 h. One colony is inoculated into 1liter BHI or BHI containing 0.1% DOC flask and shaken for 12 h at 37° C.The bacteria are then diluted to OD₆₀₀ of 0.17 and grown for 30 minfurther and then harvested as described above.

Example 18

Clostridium difficile (modified chopped meat medium with 30% glycerol)is streaked on a plate of Beef liver medium for anaerobes containing1.5% agar and cultured at 37° C. under microaerophillic conditions (5%CO₂ and 95% N₂). One colony is transferred to 1 liter of modifiedchopped meat medium or same medium containing 0.1% DOC. The bacteria arecultured under microaerophillic conditions at 37° C. for 12 h, andharvested as described above.

Example 19

Bacteroides fragilis (modified chopped meat medium with 30% glycerol) isstreaked on a modified chopped meat medium agar plate and cultured at37° C. under microaerophillic conditions (5% CO₂ and 95% N₂). One colonyis transferred to 1 liter of modified chopped meat medium or same mediumcontaining 0.1% DOC. The bacteria are cultured under microaerophillicconditions at 37° C. for 12 h, and harvested as described above.

Example 20

Yersinia pseudotuberculosis (Luria broth containing 30% glycerol) isstreaked on a Luria broth agar plate and incubated at 30° C. One colonyis transferred to 1 liter of LB and incubated for 12 h at 30° C. Thisculture is diluted (1/5) in LB or LB containing 0.1% DOC and incubated 4h at 37° C. Subsequently, the cultures are diluted in the same media toOD₆₀₀ of 0.17 and incubated a further 30 min and then harvested asdescribed above.

Example 21

Helicobacter pylori was added to BHI broth plus 4% bovine calf serum.After inoculation the flasks were flushed with 5% O₂, 10% CO₂, 85% N₂and incubated for 22 h at 37° C. with shaking. After this incubation,2.5 ml of the culture was transferred to a flask containing BHI brothwith 4% bovine calf serum or the same medium additionally containingabout 0.1% to about 0.2% bovine bile. These cultures were again flushedwith the microaerophilic gas mixture (5% O₂, 10% CO₂ 85% N₂), andincubated 20-24 h at 37° C. The cells were harvested as described above.

EXAMPLES: ENHANCED ANTIGENIC BACTERIA Example 22

Microscopic examination of wet mounted bacteria was utilized to observemotility and gross morphology. Surface layers were observed by capsulestaining in india ink (nigrosine). After air drying, the cells werecounter-stained with crystal violet. All observations were at1000×magnification.

Morphology of bacteria cultured according to methods of the presentinvention (hereinafter referred to as "ENHANCED" bacteria) was alteredcompared to those cultured in basal media alone (conventionally grown).For instance, "ENHANCED" C. jejuni aggregated, and formed large clumpsof cells, while conventionally grown cells were predominantly solitary.It was apparent from capsule staining (data not shown) that a change inthe bacterial surface was effected by culturing using methods of thepresent invention. This surface alteration actually resulted in theincreased binding by "ENHANCED" cells of the nigrosine particles fromthe stain. The "ENHANCED" bacteria remained highly motile.

Example 23

C. jejuni surface components were analyzed by phenol extraction.Extracts were made from C. jejuni 81-176 grown conventionally orcultured according to Example 2 above. C. jejuni cells were harvestedfrom culture medium by centrifugation as described above. The cellpellet was extracted for 2 h at room temperature with 1% phenol. Intactcells were separated from extracted materials by centrifugation for 45min. The supernatant containing extracted bacterial surface componentswas dialyzed against distilled water overnight. The retentate wascentrifuged 105,000×g for 3 h at 4° C. The extract pellet wasredissolved in 10% NaCl and precipitated with two volumes of cold 95%ethanol. The precipitation was repeated, and the sample was lyophilized.Subsequently, the sample was dissolved in water at 1 mg/ml for furtheranalysis.

Carbohydrate content of the extract was assayed with the generallyaccepted phenol-sulfuric acid method utilizing glucose as a standard.Uronic acid content of the extract was measured with the method ofDische using the carbazole reagent. Total protein content of the phenolextract was evaluated with the biccichinoic acid assay kit (Pierce Chem.Co., Rockford, Ill.).

It was notable that uronic acid was absent from the extracts. Manytypical bacterial capsules are composed of uronic acid polymers.Surprisingly, surface extracts of C. jejuni 81-176 were in factpredominantly protein. However, total carbohydrate content of the"ENHANCED" cell extracts was increased over cells grown conventionally.

The carbohydrate to protein ratio of the extracts is shown in Table 1below.

                  TABLE 1                                                         ______________________________________                                        Carbohydrate:Protein Ratio of Cell Surface                                    Extracts of C. jejuni Grown conventionally                                    (BHI) or According to Method of Example 2 (ENHANCED)                          Time after                                                                    addition (h)   BHI    ENHANCED                                                ______________________________________                                        1              0.02   0.02                                                    2              0.02   0.10                                                    4              0.02   0.15                                                    6              0.03   0.29                                                    ______________________________________                                    

There was a direct relationship between inclusion of DOC in the culturemedium and enhanced levels of surface extractable carbohydrate from thebacterium (Table 1). Surface extractable carbohydrates were increasedmore than 8-fold in bacteria cultured according to the methods of thepresent invention; no increase was seen in bacteria grownconventionally. The aggregation of the bacterial cells cultured in DOCmedium appeared to be attributable to the components of the surfaceextract.

Upon rehydration, the extract had a high geling capacity in waterrendering the solution highly viscous and mucus-like, which was similarin character to the aggregated bacteria. The functionality of theextract resembled mucin-like glycoproteins.

For analysis of individual monosaccharides, extracts were hydrolyzed in1 N trifluoroacetic acid in sealed vials. The samples were dried undernitrogen 2 h, and resuspended in distilled water. Sugars were separatedby HPLC using a Dionex Corp chromatographic matrix and a solvent systemconsisting of 3% of 0.5 N NaOH/97% H₂ O as solvent. Amperometricdetection was utilized for measurement of separated monosaccharides. Anauthentic monosaccharide standard composed of fucose, galactosamine,glucosamine, galactose, glucose, and mannose was also subjected to HPLCanalysis for comparison.

HPLC analysis of the hydrolyzed surface extract revealed the presence ofseveral monosaccharides (FIG. 1). There appeared to be no qualitativedifference in the carbohydrate composition of the extracts fromconventionally grown or bacteria grown according to the method ofExample 2 above. However, minor quantitative differences were apparent.

Example 24

Bacterial proteins were analyzed by SDS-PAGE and Western Blotting. Thegel system of Lugtenberg, et al. (FEBS Letters 58:254-258, 1975) wasused. The gel system is a discontinuous gel consisting of a lowacrylamide (typically 4%) stacking gel pH 6.8, and a higher percentageacrylamide separation gel pH 8.8. SDS (0.1%) was included in both gelsand all buffers used. Protein separation was according to molecularsize, and 8 or 12% acrylamide separation gels were used. Visualizationof separated proteins was by silver staining of fixed gels, andmolecular size determinations were made based on the M_(r) values ofknown proteins used as standards.

C. jejuni cell proteins were separated by SDS-PAGE and visualized bysilver staining. Four proteins including a 62 kDa protein were inducedor enhanced in cells cultured with DOC (FIG. 2).

Example 25

S. flexneri LPS was analyzed by phenol extraction. S. flexneri cellsgrown conventionally or according to the present invention asexemplified in Example 9 above were harvested from culture medium bycentrifugation as described above. Lipopolysaccharides (LPS) wereextracted by the method of Westphal and Jann (In: R. Whistler, ed.,Methods in Carbohydrate Chemistry, vol 5; p. 83, 1965). Briefly, cellscultured in BHI or as exemplified in Example 9 above were harvested bycentrifugation and washed once in PBS. The cells were then extracted for15 min at 68° C. with 45% phenol in water. The extract was cooled to 10°C. and centrifuged. The LPS-containing upper aqueous phase was aspiratedoff and dialyzed against distilled water. The retentate was centrifuged7 h at 80,000×g, 4° C. once, and three times for 3 h each at 105,000×g.The final pellet was lyophilized. Prior to analysis the LPS wasresuspended in water (1 mg/ml). The purified LPS was characterized as tocarbohydrate content as described above, and by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described belowand shown in FIG. 5.

S. flexneri protein profiles revealed no major differences betweenconventionally cultured and "ENHANCED" cells when analyzed using thisparticular SDS-PAGE system. Carbohydrate to dry weight ratios of LPSfrom "ENHANCED" cells was decreased as compared to extracts from BHIcells. However, SDS-PAGE followed by oxidation and silver staining of S.flexneri LPS demonstrated a major change in LPS structure (FIG. 5). Asseen in lane "2" of the gel, the O-antigen fraction of LPS from"ENHANCED" S. flexneri was reduced in length. This result complementedthe findings of reduced carbohydrate/dry weight ratio of LPS from"ENHANCED" cells.

These results suggest shorter O-antigen side chain presentation on"ENHANCED" cells, and potentially might render the bacteria morehydrophobic. A more hydrophobic bacterium might have greater interactionwith hydrophobic surfaces in the gut.

Example 26

Immunogenicity of proteins was determined by Western Blots. Proteinsfrom bacteria grown conventionally or according to the methods of thepresent invention as exemplified in Example 1 or 5 above were separatedby SDS-PAGE, then were electrophoretically transferred to nitrocelluloseor PVDF membranes and were blocked with a standard blocking agent [3%BSA, 50 mM Tris (pH 8.5), 50 mM NaCl, 0.2% TWEEN 20]. Primary antibodywas applied in blocking buffer, the blot was then washed and a secondaryantibody reporter cognate was applied. Following washing, the blot wasvisualized with light or chromophore producing substrates. The reportermoiety used was horse radish peroxidase or alkaline phosphatase.

FIG. 3 depicts proteins from Western Blotting with immune rabbit mucuscontaining IgA. As can be seen, the 62 kDa protein was theimmunodominant antigen. The antigenicity of the 62 kDa protein wasgreatly enhanced in cells cultured with DOC or bile. This protein wasalso the predominant antigen in the surface extract of cells culturedwith DOC.

Utilizing a mouse monoclonal antibody cross-reactive with Campylobacterflagellin, it was demonstrated that the enhanced protein was C. jejuniflagellin (FIG. 4). This wets a significant finding because severalresearchers have demonstrated that the C. jejuni flagellin is involvedin pathogenesis and associated with the invasive characteristics of thebacterium.

Example 27

Congo red dye binding was used to measure virulence. Enteric bacteriagrown conventionally (BHI) or according to the methods of the presentinvention ("ENHANCED") on BHI agar plates and containing 0.025% Congored were resuspended in distilled water and extracted with acetone for10 min. Cellular debris was pelleted by centrifugation, and the 0D₄₈₈ ofthe dye was measured with a blank solution of 40% acetone, 60% water.The dye absorbance was compared to the cell absorbance at 660 nm andexpressed as the ratio of OD₄₈₈ /OD₆₆₀. The data are shown in Table 2below.

                  TABLE 2                                                         ______________________________________                                        Congo Red Dye Binding of Enteric                                              Pathogenic Bacteria Grown Conventionally                                      (BHI) or According to Methods of the                                          Present Invention (ENHANCED)                                                                 Dye Absorbance                                                 Strain           BHI    ENHANCED                                              ______________________________________                                        C. jejuni 81-176 0.07   0.49                                                  C. jejuni 81-116 0.06   0.49                                                  S. typhimurium SR11                                                                            0.05   0.30                                                  S. flexneri 2457T                                                                              0.02   0.13                                                  V. cholera 569b  0.70   2.00                                                  ______________________________________                                    

Table 2 shows that for several species of enteric bacteria culturedaccording to methods of the present invention, Congo red dye binding wasenhanced. These results show that in vitro methods of the presentinvention are useful to induce virulence and other characteristics knownto correlate with in vivo pathogenesis for other bacterial species.

Example 28

Bacterial adhesion to cultured epithelial cells was analyzed. Bacterialadhesion was assayed as described by Galan and Curtiss (Proc. Natl.Acad. Sci. USA, 86:6383-6387, 1989). Tissue culture cells (INT-407 orHenle cells (ATCC # CCL6), and CaCo-2 (ATCC # HTB37) human intestinalcell lines) were cultured in 24-well tissue culture plates (37° C., 5%CO₂) to a confluency of 60-80%. The medium is dependent on the cell lineused, but Dulbecco's modified Eagle's medium with 10% fetal bovine serumand 50 mg/ml each of penicillin G and streptomycin was used for Henlecells, and RPMI 1640 medium with 10% fetal bovine serum and 50 mg/μleach of penicillin G and streptomycin was used for the culture of CaCo-2cells. At least 3 h before assay, the culture medium was removed and thecells were washed twice with Hank's balanced salt solution (HBSS) withmagnesium and calcium. The monolayers were then overlayed withantibiotic-free growth medium.

For adhesion assays, the bacteria were prepared as follows. For slowgrowing enteric bacteria such as Campylobacter and Helicobacter thebacterial culture density was diluted to an OD₆₂₅ of 0.1 with fresh,preequilibrated medium and then used in the assay. For Shigella andother fast growing enteric bacteria, the bacterial culture was dilutedto 0.17 at OD₆₂₅ with fresh, preequilibrated medium and then used in theassay. The bacteria were added to the epithelial cells at a multiplicityof infection of 10 bacteria per cell to avoid saturation. The number ofbacteria inoculated into the tissue culture well was calculated by platecounting. Following 2 h infection under 5% CO₂ for Campylobacter, and 30min for Shigella, unbound bacteria were removed by washing with HBSSbefore lysis of the monolayer with 0.1% deoxycholate and plating for thedetermination of adhesion.

The effect of temperature on adhesion to INT-407 cells by C. jejuni81-176 grown conventionally or according to the methods of the presentinvention as exemplified in Example 5 above is shown in Table 3 below.Differences in adhesion of several strains of C. jejuni grownconventionally or according to methods of the present invention areshown in Table 4 below.

Percent adhesion is expressed as the number of colony forming units(CFU) recovered from the monolayer divided by the number of CFUinoculated onto the monolayer multiplied by 100.

                  TABLE 3                                                         ______________________________________                                        Effect of Temperature on ADHESION to INT-407                                  Cells by C. jejuni Grown Conventionally (BHI)                                 or According to Methods of the Present                                        Invention (ENHANCED)                                                                        BHI       ENHANCED                                              Temperature   (% adhesion)                                                                            (% adhesion)                                          ______________________________________                                        37° C. 5.5       62.3                                                  42° C. 5.5       11.2                                                  ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Adhesion (FOLD INCREASE) to INT-407                                           by Different Strains of C. jejuni Grown                                       Conventionally (BHI) or According to                                          Methods of the Present Invention (ENHANCED)                                   Strain         BHI    ENHANCED                                                ______________________________________                                        81-176         1.0     8.4                                                    81-116         1.0    12.5                                                    HC             1.0    28.2                                                    ______________________________________                                    

In invasion assays, epithelial cells were grown and prepared accordingto the methods described above for the adhesion assay. Bacteria grownconventionally or according to methods of the present invention wereadded to the epithelial cells at a multiplicity of infection of 10bacteria per cell to avoid saturation. The number of bacteria inoculatedinto the tissue culture well was calculated by plate counting. Following2 h infection under 5% CO₂ for Campylobacter, and 30 min for Shigella,the infecting bacteria were aspirated off, and the monolayer wasoverlaid with growth medium containing gentamicin to kill anyextracellular bacteria. Any culturable bacteria remaining at this pointhave invaded the epithelial cell monolayer. The incubation continuedunder CO₂ for 3 h in the case of C. jejuni infected cells, and 1.5 h forShigella infected cells. Monolayers were washed with HBSS to removegentamicin, and lysed by the addition of 0.1% deoxycholate. Bacteria inthe lysates were enumerated by plate counting and percent invasion wasexpressed as the number of gentamicin resistant bacteria compared to thenumber of inoculum bacteria.

Invasion is expressed as the percent of cell entering the monolayer, asdetermined by the number of colony forming units (CFU) recovered fromthe monolayer after gentamycin treatment divided by the number of CFUinoculated onto the monolayer multiplied by 100.

The effect of temperature on invasion of INT-407 cells by C. jejuni81-176 grown conventionally or according to the methods of Example 5above is shown in Table 5 below.

Differences in invasion of INT-407 cells by several strains of C. jejunigrown conventionally or according to methods of the present inventionare shown in Table 6 below.

The effect of DOC on adhesion to and invasion into INT-407 cells by C.jejuni 81-176 grown conventionally or according to the methods ofExample 5 above is shown in Table 7 below.

The effect of DOC on adhesion to and invasion into INT-407 cells byShigella grown conventionally or according to the methods of Example 5above is shown in Table 8 below.

                  TABLE 5                                                         ______________________________________                                        Effect of Temperature on INVASION of INT-407                                  Cells by C. jejuni 81-176 Grown Conventionally                                (BHI) or According to Methods of the Present                                  Invention (ENHANCED)                                                                        BHI       ENHANCED                                              Temperature   (% invaded)                                                                             (% invaded)                                           ______________________________________                                        37° C. 2.5       49.5                                                  42° C. 4.0        7.1                                                  ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        INVASION (FOLD INCREASE) into INT-407 Cells                                   by Different Strains of C. jejuni                                             Grown Conventionally (BHI) or According                                       to Methods of the Present Invention (ENHANCED)                                Strain         BHI    ENHANCED                                                ______________________________________                                        81-176         1.0     9.2                                                    81-116         1.0    10.0                                                    HC             1.0    26.7                                                    ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Effect of DOC Concentration on Adhesion and                                   Invasion of INT-407 by C. jejuni 81-176 Grown                                 Conventionally (BHI) or According to Methods of                               the Present Invention (ENHANCED)                                              Treatment         Adhesion (%)                                                                             Invasion (%)                                     ______________________________________                                        BHI                9.3        6.3                                             ENHANCED; 0.025% DOC                                                                            18.3       17.4                                             ENHANCED; 0.1% DOC                                                                              52.6       37.0                                             ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Effect of DOC on S. flexneri Invasion (Percent)                               or Adhesion (Percent) to INT-407 Cells Grown                                  Conventionally (BHI) or According to Methods of                               the Present Invention (ENHANCED)                                              Characteristic  BHI       ENHANCED                                            ______________________________________                                        Adhesion        1.0       140.0.sup.a                                         Invasion        1.0        94.4                                               ______________________________________                                         .sup.a More than the initially added bacteria were recovered due to growt     of the bacteria during the assay                                         

Adhesion to and invasion of cultured INT-407 cells by several humanisolates of C. jejuni (i.e., 81-116, 81-176 and HC) was greatly enhancedby addition of bile or deoxycholate to the culture medium (See Tables 4and 6). The most effective dose of DOC was 0.1% (Table 7). The greatestresponse was obtained at 37° C., but not at 42° C. (the temperature atwhich Campylobacter is conventionally grown) (Tables 3 and 5).

Similar findings were made with Shigella flexneri (Table 8). Shigellagrown according to the methods of the present invention had greatlyenhanced abilities of both adhesion and invasion.

These data show methods of the present invention enhance invasion andadhesion of enteric pathogens.

Example 29

A rapid slide agglutination assay was used to show immuno-crossreactivity. C. jejuni strains grown conventionally or according to themethods of the present invention as exemplified in Example 5 wereexposed to serum IgG from animals immunized with C. jejuni 81-176 (Lior5) grown either conventionally, or according to the present invention(e.g., Example 5). The IgG antibodies were immobilized on Protein Acoated latex beads. If there are cross reactive epitopes between thetest serotype and the antibodies generated against the Lior 5 serotype,then nearly immediate clumping (i.e. agglutination) of the cells isvisible. This clumping is rated based on a scale of 0 to 3 afterallowing the reaction to proceed for a short period of time, where 0means no observable clumping and 3 means a high degree of agglutination.The results of four strains are presented in Table 9 below.

                  TABLE 9                                                         ______________________________________                                        Cross Reactivity of Lior Serotypes Grown                                      Conventionally (BHI) or According to Methods of                               the Present Invention (ENHANCED)                                                         GROWTH                                                             CULTURE    CONDITIONS  BEADS     REACTIVITY                                   ______________________________________                                        81-176 (L5)                                                                              BHI         Anti-BHI.sup.a                                                                          1                                            81-176 (L5)                                                                              BHI         Anti-ENH.sup.b                                                                          2                                            81-176 (L5)                                                                              ENHANCED    Anti-BHI  2                                            81-176 (L5)                                                                              ENHANCED    Anti-ENH  2.5                                          L2         BHI         Anti-BHI  1                                            L2         BHI         Anti-ENH  1.5                                          L2         ENHANCED    Anti-BHI  0                                            L2         ENHANCED    Anti-ENH  1.5                                          L8         BHI         Anti-BHI  2                                            L8         BHI         Anti-ENH  1                                            L8         ENHANCED    Anti-BHI  1                                            L8         ENHANCED    Anti-ENH  2.5                                          L21        BHI         Anti-BHI  0                                            L21        BHI         Anti-ENH  2                                            L21        DOC         Anti-BHI  0                                            L21        DOC         Anti-ENH  2                                            Media      BHI         Anti-BHI  0                                                       BHI         Anti-ENH  0                                            ______________________________________                                         .sup.a Antibodies induced by 81176 grown conventionally                       .sup.b Antibodies induced by 81176 grown according to methods of the          present invention as exemplified in Example 5                            

Table 9 shows all four of the tested Lior serotypes (L5, L2, L8, L21)cross-reacted with antibody generated from animals immunized withenhanced C. jejuni 81-176 Lior serotype L5. Mucus lavage from theintestines of rabbits infected with C. jejuni 81-176, comprising IgA,reacted with eight out of the 10 major clinical serotypes (i.e., humanpathogens) of C. jejuni grown according to methods of the presentinvention cross reacted with antibodies to Lior 5 serotype strain (seeTable 16 below). The results show that methods of the present inventionsignificantly extend the number of Lior serotypes which cross react withanti-serum from animals immunized with Lior 5 serotype strain of C.jejuni.

Also, several species of Shigella grown according to methods of thepresent invention, but not those grown conventionally, cross react withIgG antibodies from animals immunized with Shigella flexneri 2457T grownwith DOC.

EXAMPLES: VACCINE EFFICACY Example 30

The ferret model for studying Campylobacter pathogenesis can be used asa model to evaluate vaccine efficacy in protecting against colonizationand/or disease because infection of ferrets reproducibly generates twoof the three disease manifestations seen in humans.

Twenty-four 7- to 9-week-old male ferrets were immunized orally witheither PBS (as control), or formalin-fixed C. jejuni strain 81-176 grownconventionally (BHI) or according to methods of Example 5 (ENHANCED).Serum was isolated to determine baseline IgG titres. All vaccines andPBS were administered in the presence of the adjuvant LT, two times, oneweek apart (Day 0 and day 7, "vaccination"). Serum was collected oneweek later (day 14) ("post-vaccination") to determine IgG antibodytitres. Four weeks post-vaccination (challenge), the ferrets wereanesthetized with ACE promazine-Ketamine and challenged orally with a 10ml PBS solution containing live C. jejuni 81-176 (1×10¹⁰ CFU).Thereafter the animals were monitored daily for mucoid diarrhea,bacteremia, fecal shedding of Campylobacter, weight changes, occultblood, and fecal leukocytes. Bacteremia was detected by drawing 1 to 2ml of blood from the jugular vein of anesthetized ferrets and incubatingthe specimen in a vented trypticase soy broth culture. Subcultures toblood agar plates were taken at 2, 5 and 7 days post challenge. Serumsamples were collected prior to immunization (baseline), one week afterthe second immunization, and at the time of challenge, and one week postchallenge to determine IgG titres.

Occult blood was detected by testing fecal material on a Hemacult card.Fecal material was smeared on a slide and stained with methylene blue todetect fecal leukocytes. Fecal shedding of Campylobacter was establishedby culturing smears from rectal swabs on Campylobacter-selective mediumplates (trypticase soy agar, 5% sheep blood, trimethoprim, vancomycin,polymyxin B, cephalothin, and amphotericin B, Remel, Lenexa, KS).Results from these experiments are presented in Tables 10 and 11 below.

                  TABLE 10                                                        ______________________________________                                        Vaccination Protects Against                                                  C. jejuni 81-176 in Ferrets                                                                POSITIVE                                                                      COLONIZATION                                                                  DAY 5                                                            VACCINE      POST-CHALLENGE.sup.a                                                                        DISEASE.sup.b                                      ______________________________________                                        PBS          6/6           1/6                                                BHI          0/6           2/6                                                ENHANCED     0/5.sup.c     0/6                                                ______________________________________                                         .sup.a Number positive colonization/number tested                             .sup.b Presented with green mucus/unformed/watery stools                      .sup.c One animal died in the group after disease state was determined bu     before colonization was scored                                           

                  TABLE 11                                                        ______________________________________                                        Ferret Sera IgG Geometric Mean Titer                                                             One week                                                                      post-    At     One week post                              Group    Baseline  vaccine  challenge                                                                            challenge                                  ______________________________________                                        PBS      6.4        4.9       6.4  1380.4                                     BHI      6.4       94.0      94.0  26505.3                                    ENHANCED 6.8       234.4    1621.8 56234.1.sup.a                              ______________________________________                                         .sup.a Mean titre for the five surviving animals                         

Table 10 shows that upon live challenge, animals immunized with akilled-whole cell vaccine of the present invention were protectedagainst colonization and disease. The data in Table 11 show that a muchgreater IgG antibody titre results from vaccines of the presentinvention (ENHANCED) than from enteric bacteria grown conventionally(BHI).

These results demonstrate that immunogenicity (Table 11) and protectionfrom infection (Table 10) was obtained with vaccines of the presentinvention and was greater than that seen when animals were vaccinatedwith bacteria grown conventionally. Therefore bacteria produced by themethods of the present invention are useful as vaccines to protectmammals from infection.

Example 31

Mice do not naturally develop Campylobacter or Shigella infections as doferrets, but they have been used by those skilled in the art to showresistance to intestinal colonization upon oral challenge of immunizedanimals or resistance to illness via lung infection of immunizedanimals. The mouse intranasal inoculation model then can be used topredict the efficacy of vaccines for use in other animals or humans.This assay was described by Mallet, et al. (Vaccine, 11:190-196, 1993).

Groups of 10 female Balb/c mice about sixteen weeks old were immunizedorally with phosphate-buffered saline (PBS) C. jejuni conventionallygrown (BHI) or C. jejuni crown according to Example 5 (ENHANCED) indoses of about: 10⁷ CFU or 10⁹ CFU, then challenged. IgA titres fromintestinal mucus in each group were determined by ELISA methods and arepresented in Table 12 below.

                  TABLE 12                                                        ______________________________________                                        IgA Responses After Oral Immunization with (10.sup.7                          or 10.sup.9) Campylobacter Whole Cell Vaccines in Mice                                        Lavage   %                                                    Immunization    IgA Titre.sup.a                                                                        Responders.sup.b                                     ______________________________________                                        PBS             23       14                                                   ENHANCED (10.sup.7)                                                                           114      75                                                   ENHANCED (10.sup.9)                                                                           78       75                                                   BHI (10.sup.7)  40       25                                                   BHI (10.sup.9)  32       12                                                   ______________________________________                                         .sup.a Lavage titre indicates the mean antiC. jejuni IgA titre obtained       for each individual group of mice.                                            .sup.b Responders are defined as those animals whose endpoint titres          exceeded 2 standard deviations above the mean of the animals receiving PB     alone                                                                    

Table 12 shows that animals immunized with bacteria grown according tothe methods of the present invention have a higher intestinal IgAantibody titre as presented by a greater percentage of responders thananimals immunized with bacteria grown conventionally.

EXAMPLE: MECHANISM OF ANTIGENIC ALTERATION OR ENHANCEMENT BY DOC Example32

Although not intending for the present invention to be limited to anyparticular mechanism of action, the present inventors have obtainedevidence that suggests that deoxycholate (DOC) appears to have a twofold action in altering or enhancing the antigenicity of entericbacteria. Evidence indicates that one aspect of DOC's effect is mediatedthrough calcium dependent effects, as DOC binds calcium and thus lowersthe calcium concentration in the medium. The evidence is as follows.When C. jejuni 81-176 is cultured with the membrane permeable calciumchelator BAPTA/AM and without DOC, its invasiveness of INT-407 cells isenhanced approximately 10-fold (see Table 13 below). BAPTA/AM treatmentalone, however, does not enhance the immuno-cross reactivity of C.jejuni 81-176 cells.

The results shown in Table 13 were obtained using C. jejuni 81-176cultured according to the protocol described in Example 5 except that 25μM BAPTA/AM was substituted for 0.1% DOC. The invasion assays werecarried out and scored as described in Example 28 in Section 7 above.

                  TABLE 13                                                        ______________________________________                                        Invasion of INT-407 Cells by C. jejuni Grown                                  Conventionally (BHI) or with BAPTA/AM                                                        Culture condition                                              Strain           BHI    BAPTA/AM                                              ______________________________________                                        C. jejuni 81-176 3.0    36.9                                                  ______________________________________                                    

Several bacterial genera that are susceptible to antigenic enhancementor alteration by bile or bile salts such as DOC (e.g., Campylobacter,Shigella, Helicobacter) have genes homologous to low calcium response(lcr) genes from Yersinia. The lcr locus is known to regulate virulenceof Yersinia in response to low calcium levels. Two Campylobacter genesinvolved in flagellin expression and assembly which are required forinvasion (flaA, flbA) are regulated in part by the lcr product. Analysisof the behavior of Campylobacter flaA and flbB mutants grownconventionally or under virulence enhancing conditions of the presentinvention show that invasion, but not Congo red dye binding or increasedcross-reactivity, is calcium dependent (see Table 14 below).

The results shown in Table 14 were obtained using C. jejuni culturedconventionally or according to the methods of the present invention asexemplified in Example 5. The invasion assays were carried out andscored as described in Example 28 above.

                  TABLE 14                                                        ______________________________________                                        Invasion of INT-407 Cells by C. jejuni Mutants                                Grown Conventionally (BHI) or According to                                    Methods of the Present Invention (ENHANCED)                                                  Culture condition                                              Strain           BHI    BAPTA/AM                                              ______________________________________                                        C. jejuni 81-176 3.0    36.9                                                  C. jejuni flaA   0.05   0.05                                                  C. jejuni flbA   0.01   0.04                                                  ______________________________________                                    

The C. jejuni fla and flbA mutants do exhibit significantly enhancedCongo red binding and immuno-cross reactivity when cultured with DOC(see Table 15 and FIG. 6, respectively). The results shown in Table 15and FIG. 6 were obtained using C. jejuni cultured conventionally oraccording to the methods of the present invention as exemplified inExample 5 above. The Congo red dye binding assays, whose results areshown in Table 15, were carried out as described in Example 26 above.The immuno-cross reactivity, whose results are shown in FIG. 6, werecarried out as described in Example 29 above.

                  TABLE 15                                                        ______________________________________                                        Congo Red Dye Binding by C. jejuni Mutants                                    Grown Conventionally (BHI) or According to                                    Methods of the Invention (ENHANCED)                                                          Culture condition                                              Strain           BHI    ENHANCED                                              ______________________________________                                        C. jejuni 81-176 0.07   1.68                                                  C. jejuni flaA   0.10   1.60                                                  C. jejuni flbA   0.12   0.80                                                  ______________________________________                                    

The flaA mutant is unable to express flagellin. The flaA mutant and aflaA-flaB double mutant (received from C. Grant, NIH) are bothnoninvasive even after treatment with DOC, indicating that flagellin isrequired for invasion. Interestingly, the normally exhibited (i.e.,non-DOC induced) immuno-cross reactivity observed between the isogenicparent strain of these fla mutants and certain other Lior serotypes ofC. jejuni is absent in the mutants. However, DOC treatment can inducethese flagellin-less mutants to exhibit enhanced immuno-cross reactivityand Congo red binding.

These findings indicate that DOC regulates virulence functions inenteric bacteria via calcium-dependent (e.g., invasiveness) andcalcium-independent (e.g., Congo red dye binding) mechanisms. Thesefindings further suggest that enhanced immuno-cross reactivity and Congored binding induced by DOC is at least in part flagellin-independent.

EXAMPLE: DOC INDUCES ENHANCED SEROTYPE AND SPECIES IMMUNO-CROSSREACTIVITY OF CAMPYLOBACTER JEJUNI Example 33

The serotype immuno-cross reactivity of Campylobacter jejuni grownaccording the methods of the present invention was examined using therapid slide agglutination assay. The assay used intestinal mucus fromimmunized and non-immunized rabbits to determine the effects of alteringculture conditions on the cross-reactivity of heterologous strains ofCampylobacter. The rabbits were immunized with live C. jejuni 81-176grown conventionally. The agglutination activity of the mucus antibodieswere tested against twenty-four Campylobacter strains, comprisingeighteen serotypes, grown conventionally in BHI-YE medium or accordingto the methods of the invention as exemplified in Example 5.

The results of the agglutination assays show that thecross-agglutination of heterologous Campylobacter strains was broaderand, in many cases, stronger when the strains were grown according tothe methods of the invention than when they were grown conventionally.Specifically, there was an over two fold increase in heterologousagglutination reactivity: 6 of 24 conventionally-grown heterologousstrains agglutinated at level+or greater in the anti-81-176 immunemucus, whereas 14 of the same 24 strains grown under DOC conditionsagglutinated at level +or greater. Further, while eighteen of theheterologous strains demonstrated weak (±) or no agglutination whenconventionally grown, eleven of these same strains showed an enhancedagglutination response when grown under ENHANCED culture conditions(e.g., DOC containing medium).

Table 16 illustrates cross-reactivity of anti-81-176 immune rabbit mucusagainst 19 heterologous Lior serotypes consisting of 22 strains grownconventionally (BHI-YE) or using the methods of Example 5 (ENHANCED).Even though this experiment assayed only a fraction of the known Liorserotypes, the results demonstrate that the methods of the inventioninduce substantial immuno-cross reactivity between Lior serotypes. Theresults further show that the DOC enhanced or induced antigens inCampylobacter appear to be important in the secretory IgA responseassociated with resistance to and recovery from intestinal infection byCampylobacter.

It should be further noted that strains of Lior serotype 8 are of adifferent species, Campylobacter coli. One of the 2 strains (VC167) ofthis serotype strongly agglutinated (3+) in anti-81-176 immune rabbitmucus. This result indicates that a vaccine derived from a C. jejunistrain (e.g., Lior 5), not only can cross-protect against heterologousserotypes within the same species, but also other Campylobacter species(e.g., Campylobacter coli). Also worth noting is that Lior serotypes 1,2, 4, 9, and 11 are among the most prevalent disease-associatedserotypes world-wide. They all demonstrated detectable cross-reactivityin this assay.

                  TABLE 16                                                        ______________________________________                                        Agglutination Response of 20 Campylobacter                                    Serotypes Grown Conventionally (BHI-YE) or                                    According to the Methods of the Invention                                     (ENHANCED) to Non-immune.sup.b or Anti-81-176.sup.a                           Immune Rabbit Mucus                                                                    Agglutination response.sup.c                                         Lior       Non-immune mucus                                                                             Immune Mucus                                        Strain                                                                              Serotype BHI-YE  ENHANCED BHI-YE                                                                              ENHANCED                                ______________________________________                                        134    1       -       -        -     ++                                      195    2       -       -        -     ±                                    1      4       -       -        -     +++                                     170    5       -       -        +++   ++++                                    81-176                                                                               5       -       -        ++++  ++++                                    6      6       -       -        ++++  +++                                     81-116                                                                               6       -       -        +     ++                                      35     7       -       -        -     +                                       52     8       -       +        +     ++                                      VC-167                                                                               8       -       -        +     +++                                     VC-159                                                                               8       -       -        ±  -                                       88     9       -       -        -     ±                                    244   11       -       -        ±  +++                                     556   17       -       -        +     -                                       563   18       -       -        -     -                                       544   19       -       ++       -     ++                                      699   21       -       -        ±  ++                                      1180  28       -       -        +     +++                                     1982  29       -       -        -     -                                       910   32       -       ++       -     ++                                      2074  36       -       -        -     -                                       HC    36       -       +        -     +                                       2984  46       -       -        -     -                                       79171 72       -       -        -     -                                       ______________________________________                                         .sup.a The anti81-176 mucus were obtained from rabbits infected with live     C. jejuni 81176 grown conventionally                                          .sup.b The nonimmune mucus were obtained from uninfected rabbits              .sup.c The agglutination responses range from negative (-), to very weak      (±), to very strong (++++)                                            

EXAMPLE: ADDITIONAL EXPERIMENTS OF VACCINE EFFICACY OF CAMPYLOBACTERExample 34

The protective efficacy of formalin-fixed whole cell Campylobacterjejuni grown according to the methods of the present invention wasdetermined using the mouse colonization model reported by Baqar (Infect.& Immun., 15 63:3731-3735, 1995).

C. jejuni 81-176 was grown and harvested according to Example 5 andinactivated with 0.075% formalin as described above. Groups of five 6 to8 week old female Balb/c mice were administered three oral doses (0.25ml/dose in endotoxin-free PBS) containing either 10⁵, 10⁷, or 10⁹inactivated bacterial particles alone or in combination with 25 μg ofthe heat labile enterotoxin from E. coli (LT). Doses were given at 48hour intervals and immediately after two 0.5 ml doses of 5% sodiumbicarbonate solution (pH 8.5) were given at 15 minute intervals, toneutralize gastric acidity. As controls, groups of animals werevaccinated with PBS alone or in combination with the LT adjuvant.Approximately 28 days after administration of the third dose, vaccinatedanimals were challenged either nasally or orally with approximately 10⁸colony forming units (CFU) of live conventionally grown C. jejuni81-176. The duration of intestinal colonization was determined bymonitoring fecal shedding every day over a 9 day period. Fecal materialwas emulsified in sterile PBS and aliquots plated on Campylobacter bloodagar. Plates were incubated at 35° C. under microaerophillic conditions(Campylobacter GasPak, BBL) for 3-5 days to allow growth of C. jejuni.Colonization results are expressed as the percentage of animals sheddingCampylobacter organisms on a given sample day.

As shown in FIG. 7, all nasally challenged animals, both immunized andcontrol, shed organisms immediately after challenge (day 1). Eighty toone hundred percent of the control animals remained colonized for 9 daysafter challenge. In contrast, significantly fewer animals in thevaccinated groups shed organisms during the course of the 9 day assayperiod. Both the degree of and time to clearance of challenge organismswere dependent on the amount of vaccine administered. The low (10⁵particles/dose) and intermediate vaccine doses (10⁷ particles/dose) gavea gradual and incomplete rate of clearing. The presence of adjuvantincreased the degree of protection at these doses. Surprisingly, at thehighest dose tested (10⁹ particles/dose) the non-adjuvantized vaccineproduced a level of protection equal to or slightly greater than thatobtained when a comparable dose was administered with the LT adjuvant.

Similar results were obtained when orally vaccinated animals weresubsequently challenged orally (see FIG. 8). These results indicate thatimmunization with inactivated Campylobacter grown according to themethods of the present invention affords protection against subsequentchallenges of live Campylobacter and the immunization is efficaciouseven when administered orally without the use of an adjuvant.

The protective efficacy of formalin-fixed whole cell Campylobacterjejuni grown according to the methods of the present invention (see,e.g., Example 5) administered intraperitoneally (IP) was also evaluated.For these experiments, groups of 20 female Balb/c mice were administereda single dose of 1.3×10¹⁰, 2.5×10⁹, 5.0×10⁸, 1.0×10⁸ or 2.0×10⁷inactivated C. jejuni particles in 0.5 ml endotoxin-free PBS withoutadjuvant. The animals were challenged 14 days later with a single lethaldose of live C. jejuni 81-176 (approximately 1.0×10¹⁰ CFU inendotoxin-free PBS) delivered intraperitoneally. Animals were monitoreddaily for 4 days for mortality.

As shown in Table 17, a single intraperitoneal dose of 5.0×10⁸inactivated C. jejuni particles induced an immunologcal responsesufficient to protect animals against a live C. jejuni challenge.

                  TABLE 17                                                        ______________________________________                                        Protection Afforded By IP Delivered                                           V2Inactivated C. jejuni Prepared According to                                 the Methods of the Present Invention                                                  Mortality                                                                     Day                                                                   Dose      1         2     3      4   Survivors                                ______________________________________                                        1.3 × 10.sup.10                                                                   0         4     0      0   16                                       2.5 × 10.sup.9                                                                    0         0     0      0   20                                       5.0 × 10.sup.8                                                                    0         0     0      0   20                                       1.0 × 10.sup.8                                                                    11        7     0      0    2                                       5.0 × 10.sup.7                                                                    10        6     2      0    2                                       PBS Control                                                                             4         3     2      0    1                                       ______________________________________                                    

EXAMPLES: DOC ENHANCED INVASIVENESS, CONGO RED BINDING AND IMMUNO-CROSSREACTIVITY OF SHIGELLA Example 35

The invasiveness of Shigella sp. grown in vitro is affected by theculture's growth phase. The invasiveness of Shigella flexneri 2457Tcells grown conventionally (BHI), or according to the methods of theinvention as exemplified by Example 9 (DOC-EL) (wherein the cells arefrom an early log phase culture), or according to Example 9 but allowingthe culture to reach late log phase before harvesting the cells (DOC-LL)was tested according to the procedures described in Example 28. Theresults show that culturing with DOC enhances invasiveness and that themaximum enhancement is achieved during early log phase of growth (seeFIG. 9).

Culturing with DOC according to the methods of the invention alsoenhances the invasiveness of other Shigella species, S. sonnei, and S.dysentariae (see FIG. 10). In polarized epithelial cells the enhancedinvasiveness of Shigella was observed only when the epithelial cellswere infected basolaterally by the bacteria. This finding is consistantwith the invasion process observed in vivo.

Comparitive studies show that Shigella grown according to the methods ofthe present invention are nearly 10-fold more invasive than Shigellaprepared according to the procedure described by Pope et al. (Infect. &Immun., 63:3642-3648 1995).

Example 36

Shigella cultured according to the methods of the present invention alsoexhibit enhanced Congo red binding. S. flexneri 2457T and S. sonneigrown conventionally (BHI) or according to the methods of the presentinvention as exemplified in Example 9 were assayed for their dye bindingabilities using procedures described in Example 26 above. The resultsshow that growth in DOC enhanced Congo red binding by the two Shigellaspecies 10 to 20 fold (see Table 18).

                  TABLE 18                                                        ______________________________________                                        Congo Red Binding by S. flexneri and S. sonnei                                Grown Conventionally (BHI) or According to                                    Methods of the Present Invention (ENHANCED)                                                  Culture condition                                              Strain           BHI    ENHANCED                                              ______________________________________                                        S. flexneri 2457T                                                                              0.04   0.44                                                  S. sonnei        0.02   0.40                                                  ______________________________________                                    

Shigella is divided into four species and various serotypes. Theimmuno-cross reactivity of Shigella flexneri grown according the methodsof the present invention was examined using the agglutination assay asdescribed in Example 28. The assay used antiserum from immunized rabbitsto determine the effects of culture conditions on the immuno-crossreactivity of different Shigella species. The rabbits were immunizedwith formalin-fixed Shigella flexneri 2457T grown according to themethods of Example 9. The agglutination activity of the IgG antibodiesobtained from the immunized animals were tested against all fourShigella species grown conventionally in BHI medium or according to themethods of the invention (e.g., Example 9). The results of theagglutination assays show that growth with DOC significantly enhancedthe agglutination activity of the homologous Shigella flexneri as wellas those of the three heterologous Shigella species to anti-S. flexneriantibody (see FIG. 11).

EXAMPLE: VACCINE EFFICACY OF SHIGELLA Example 37

The protective efficacy of formalin-fixed whole cell Shigella flexnerigrown according to the methods of the present invention was determinedusing the mouse nasal challenge model developed by C. P. Mallett et al.(Vaccine, 11:190-196, 1993). Briefly, Shigella flexneri was grown andharvested according to the methods exemplified in Example 9 andinactivated with 0.075% formalin as described in Example 30.Approximately 10⁷ inactivated bacterial particles were used to vaccinate14-16 week-old female Balb/c mice. The inactivated S. flexneri wassuspended in sterile, endotoxin-free PBS at a concentration of 10⁸particles/ml and 35 μl of this material was administered nasally togroups of 10 lightly anesthetized animals. A total of threeimmunizations were given at 14 day intervals.

To test the effect of an adjuvant on the protective ability of theShigella whole cell vaccine, groups of animals were immunized using asuspension that contained the inactivated vaccine and 5 μg of theheat-labile enterotoxin from E. coli (LT). Fourteen days after the thirdimmunization, animals were challenged nasally with a sublethal wastingdose (10⁵ cfu) of either live S. flexneri or S. sonnei. Immediatelybefore and at 1, 2, 5, and 7 days following the challenge, animals wereweighed and the mean group weight determined. Results are shown in Table19.

                  TABLE 19                                                        ______________________________________                                        Inactivated Shigella flexneri Whole Cell                                      Vaccine Protects Mice Against Nasal Challenge                                 With Live S. flexneri or S. sonnei                                                              Percent Weight change                                       Challenge         Post Challenge                                              organism                                                                              Vaccination                                                                             Day 1   Day 2  Day 5 Day 7                                  ______________________________________                                        S. flexneri                                                                           PBS       -8.1    -18.2  -17.7 -18.3                                          vaccine   -5.4     -2.9  -2.5  -1.6                                           vaccine + -7.0     -2.0  -1.5   1.5                                           adjuvant                                                              S. sonnei                                                                             PBS       -8.1    -17.5  -9.3  -6.4                                           vaccine   -6.7    -11.3  -6.5  -6.0                                           vaccine + -6.4     -5.2  -1.3  -2.1                                           adjuvant                                                              ______________________________________                                         Ten mice in each group were immunized 3X nasally with vaccine comprsing       10.sup.7 inactivated S. flexneri grown according to Example 9            

The mice immunized with vaccine comprising inactivated S. flexneri grownaccording to the methods of the invention were protected againstchallenge with live S. flexneri organisms. Those mice suffered lessweight loss and underwent more rapid weight recovery as compared tounvaccinated mice, i.e. the PBS sham control group. Surprisingly, the S.flexneri vaccine also protected the mice against challenge with live S.sonnei. Interestingly, animals receiving the vaccine alone without theLT adjuvant were as well protected against the homologous S. flexnerichallenge as animals that received the adjuvantized vaccine. The S.flexneri vaccine alone also conferred protection to heterologouschallenges by S. sonnei. The inclusion of the LT adjuvant, however,noticibly enhanced the protection against the challenge by S. sonnei.These findings indicate that vaccine comprising inactivated Shigellaprepared according to the methods of the invention is effective in arecognized Shigella disease model and does not require an adjuvant inpreventing or attenutating various Shigella infections.

EXAMPLE: FACTORS THAT AFFECT THE ADHESIVENESS OF H. PYLORI TO ANIMALCELLS Example 38

The adherence of H. pylori is enhanced by growth in glycocholate orbile. Cells of H. pylori strain NB3-2 or G1-4 were added to BHI brothplus 4% bovine calf serum. After inoculation the flasks were flushedwith 10% CO₂ -5%O₂ -85%N₂ and incubated for 22 h at 37° C. with shaking.After this incubation, the culture was diluted 1 to 10 to a flaskcontaining 1 liter of the BHI medium with 4% bovine calf serumcontaining various concentrations of bovine bile (0.025% to 0.2%). Thesecultures were again flushed with the same gas mixture, and incubated at37° C. The cells were harvested at various times up to 18 h and theiradherence to INT-407 cells assayed using the methods described inExample 28. The results show that culturing with bile enhanced theadhesiveness of H. pylori to INT-407 cells (see FIGS. 12 and 13). Forthe NB3-2 strain, peak adhesiveness, a 4 to 6 fold increase over that ofnon-enhanced culture, occurred after about 8 h of growth (FIG. 12). Forthe G1-4 strain, peak adhesiveness, a 2 to 3 fold increase over that ofnone-enhanced culture, occurred between 12-14 h of growth in 0.2% bile(FIG. 13). These "peak" times generally corresponded to the period whenthe culture of each strain was in log phase growth.

EXAMPLE: VACCINE EFFICACY OF HELICOBACTER GROWN ACCORDING TO THE METHODSOF THE PRESENT INVENTION Example 39

The protective efficacy of formalin-fixed whole cell Helicobacter pylorigrown according to the methods of the present invention was determinedusing the mouse Helicobacter felis gastric colonization model describedby Chen et al. (Lancet, 339:1120-1121, 1992). Helicobacter pylori strainG1-4 was grown as a seed culture for about 22 h at 37° C. under 10% CO₂,90% air in BHI media containing 4% bovine calf serum. An aliqout of thisculture was used to inoculate a 10-fold volume of the same mediacontaining 0.1% (v/v) bovine bile. After 12-14 h of growth at 37° C.,the cells are harvested by centrifugation and resuspended in 1/10 of theoriginal volume of Hank's Balanced Salts Solution (HBSS) at roomtemperature. Cells were recentrifuged and again suspended in 1/100 ofthe original volume of HBSS. To the buffered cell suspension, formalinwas added to a concentration of 0.075% and the cells inactivated bystirring the suspension at room temperature for 6 h then cooling thesolution at 4° C. for 18 hours.

Protection potential was routinely measured by administering 3 doses ofthis inactivated whole cell vaccine orally to 6-8 week old female Balb/cHelicobacter-free mice at days 0, 7 and 14 or at days 0, 7 and 21. Dosesof 10⁹ bacterial particles per dose were evaluated in combination withthe heat labile enterotoxin of E. coli. Fourteen days after the thirdimmunizing dose, animals were challenged orally with a single dose (10⁷CFU/dose) of live H. felis.

Two weeks after challenge the animals were sacrificed and antral stomachsegments analyized for urease activity to determine the presence of H.felis. Urease activity was determined by incubating antral tissuesamples in 0.5 ml Stuart's Urease Broth (Remel) at room temperature for4-24 hours. A color change from clear to red occuring within this periodwas taken as a positive urease result.

As shown in Table 20, administration of enhanced Helicobacter whole cellvaccine prepared using H. pylori strain G1-4 protected animals againstan H. felis oral challenge.

                  TABLE 20                                                        ______________________________________                                        Protection Against Helicobacter Infections with                               Vaccines Comprising Inactivated H. pylori Grown                               According to Methods of the Present Invention                                            Challenge                                                          Immunizing Organisms    Colonized/                                                                             Percent                                      Agents.sup.a                                                                             (10.sup.7 CFU)                                                                             Total    Protection                                   ______________________________________                                        Experiment 1                                                                  H. pylori.sup.b                                                                           H. felis     4/13    71                                           PBS + LT   H. felis     9/9       0                                           Experiment 2                                                                  H. pylori.sup.b                                                                           H. felis     2/15    87                                           PBS + LT   H. felis     10/10     0                                           ______________________________________                                         .sup.a All agents given with 10 μg LT (Labile toxin of ETEC) adjuvant      as 3 oral doses at 7 day intervals                                            .sup.b Given as 1 × 10.sup.9 CFU of Strain G14 in a 0.25 ml dose   

Results of these experiments show the relevance of enhanced entericbacterial properties to in vivo immunogenicity.

The methods of the present invention produce bacteria capable ofinducing an immunogenic response which is protective and therefore areuseful as vaccines.

DEPOSIT OF MICROORGANISM

The following microorganisms have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA, andhave the indicated accession numbers:

    ______________________________________                                        Microorganism  Accession No.                                                                            Deposit Date                                        ______________________________________                                        Helicobacter pylori NB3-2                                                                    ATCC 55714 September 29, 1995                                  Helicobacter pylori G1-4                                                                     ATCC 55713 September 29, 1995                                  ______________________________________                                    

Other equivalents of the methods of the present invention may be easilydetermined by those skilled in the art and such equivalents are intendedto be included by this invention. The foregoing disclosure includes allthe information deemed essential to enable those skilled in the art topractice the claimed invention. Because the cited patents orpublications may provide further useful information all the materialscited herein are hereby incorporated by reference in their entirety.

What is claimed is:
 1. A method for detecting antibodies to Shigellabacteria in an animal or biological sample therefrom, comprising thesteps of:a) contacting said biological sample with a Shigella bacteriumor a fragment thereof having an enhanced antigenic property, whereinsaid Shigella bacterium is harvested from a culture of a Shigellaspecies, wherein said culture is at about early log phase, between earlylog phase and stationary phase, or at about stationary phase, grown invitro under a combination of conditions comprising:1) about 0.05% toabout 3% bile or about 0.025% to about 0.6% of one or more bile acids orsalts thereof; 2) at a temperature between about 30° C. and about 42°C.; 3) in air or a gas mixture, wherein the gas mixture comprises i)about 5% to about 20% CO₂ with about 80% to about 95% air; ii) about 5%to about 20% CO₂ with about 80% to about 95% N₂ ; or iii) about 5% toabout 10% O₂ with about 10% to about 20% CO₂ with about 70% to about 85%N₂ ; and 4) a divalent cation chelator selected from the groupconsisting of 0 to about 100 μM of BAPTA/AM, 0 to about 10 mM of EGTA,and 0 about 100 μM of EGTA/AM, and b) detecting an antibody present insaid biological sample binding to the Shigella bacterium or fragmentthereof wherein said detecting is by means of an immunoassay.
 2. Themethod according to claim 1, wherein said bile acid or salt thereofcomprises deoxycholic acid or deoxycholate.
 3. The method according toclaim 1, wherein said Shigella bacterium is a Shigella species selectedfrom the group consisting of flexneri, sonnei, dysentariae and boydii.4. The method according to claim 3, wherein said Shigella bacterium isShigella dysentariae.
 5. The method of claim 1, wherein said immunoassayis a radioimmunoassay, enzyme-linked immunosorbent assay (ELISA),flourescent immunoassay, or fluorescence polarization immunoassay(FPIA).
 6. A method for detecting antibodies to Shigella bacteria in ananimal or biological sample therefrom, comprising the steps of:a)contacting said biological sample with a Shigella bacterium or afragment thereof having an enhanced antigenic property, wherein saidShigella bacterium is harvested from a culture of a Shigella species,wherein said culture is at about early log phase, between early logphase and stationary phase, or at about stationary phase grown in vitrounder a set of conditions comprising:1) a divalent cation chelatorselected from the group consisting of about 1.0 to about 25 μM ofBAPTA/AM, about 0.5 to about 10 mM of EGTA, and about 1 to about 100 μMof EGTA/AM; 2) at a temperature between about 30° and about 42° C.; and3) in air or an microaerophilic condition, wherein the microaerophiliccondition comprises i) about 5% to about 20% CO₂ with about 80% to about95% air; ii) about 5% to about 20% CO₂ with about 80% to about 95% N₂ ;or iii) about 5% to about 10% O₂ with about 10% to about 20% CO₂ withabout 70% to about 85% N₂ ; and b) detecting an antibody present in saidbiological sample binding to the Shigella bacterium or fragment thereofwherein said detecting is by means of an immunoassay.
 7. The method ofclaim 6, wherein said immunoassay is a radioimmunoassay, enzyme-linkedimmunosorbent assay (ELISA), flourescent immunoassay, or fluorescencepolarization immunoassay (FPIA).
 8. The method according to claim 6wherein said Shigella bacterium is Shigella flexneri.
 9. The methodaccording to claim 8 wherein said Shigella flexneri is strain 2457T andthe combination of conditions comprises:1) about 0.1% bile salt which isdeoxycholate or about 0.8% bile; 2) the temperature is about 37° C.; 3)in air; andthe culture is in early log phase.
 10. A method for detectingShigella bacteria in an animal or biological sample therefrom,comprising the steps of:a) contacting said biological sample with anantibody that binds the Shigella bacteria, or a fragment thereof, saidantibody having been obtained using a Shigella bacterium having anenhanced antigenic property, wherein said Shigella bacterium having anenhanced antigenic property is harvested from a culture of a Shigellaspecies, wherein said culture is at about early log phase, between earlylog phase and stationary phase, or at about stationary phase, grown invitro under a combination of conditions comprising:1) about 0.05% toabout 3% bile or about 0.025% to about 0.6% of one or more bile acids orsalts thereof; 2) at a temperature between about 30° C. and about 42°C.; 3) in air or a gas mixture, wherein the gas mixture comprises i)about 5% to about 20% CO₂ with about 80% to about 95% air; ii) about 5%to about 20% CO₂ with about 80% to about 95% N₂ ; or iii) about 5% toabout 10% O₂ with about 10% to about 20% CO₂ with about 70% to about 85%N₂ ; and 4) a divalent cation chelator selected from the groupconsisting of 0 to about 100 μM of BAPTA/AM, 0 to about 10 mM of EGTA,and 0 about 100 μM of EGTA/AM, and b) detecting an antibody binding tothe Shigella bacteria or fragment thereof present in said biologicalsample, wherein said detecting is by means of an immunoassay.
 11. Themethod of claim 10, wherein said immunoassay is a radioimmunoassay,enzyme-linked immunosorbent assay (ELISA), flourescent immunoassay, orfluorescence polarization immunoassay (FPIA).
 12. The method accordingto claim 10, wherein said bile acid or salt thereof comprisesdeoxycholic acid or deoxycholate.
 13. The method according to claim 10,wherein said Shigella bacterium is a Shigella species selected from thegroup consisting of flexneri, sonnei, dysentariae and boydii.
 14. Themethod according to claim 13, wherein said Shigella bacterium isShigella flexneri.
 15. A method for detecting Shigella bacteria in ananimal or biological sample therefrom, comprising the steps of:a)contacting said biological sample with an antibody that binds theShigella bacteria, or a fragment thereof, said antibody having beenobtained using a Shigella bacterium having an enhanced antigenicproperty, wherein said Shigella bacterium having an enhanced antigenicproperty is harvested from a culture of a Shigella species wherein saidculture is at about early log phase, between early log phase andstationary phase, or at about stationary phase, grown in vitro under aset of conditions comprising:1) a divalent cation chelator selected fromthe group consisting of about 1.0 to about 25 μM of BAPTA/AM, about 0.5to about 10 mM of EGTA, and about 1 to about 100 μM of EGTA/AM; 2) at atemperature between about 30° and about 42° C.; and 3) in air or anmicroaerophilic condition, wherein the microaerophilic conditioncomprises i) about 5% to about 20% CO₂ wits about 80% to about 95% air;ii) about 5% to about 20% CO₂ with about 80% to about 95% N₂ ; or iii)about 5% to about 10% O₂ with about 10% to about 20% CO₂ with about 70%to about 85% N₂ ; and b) detecting an antibody binding to the Shigellabacteria or fragment thereof present in said biological sample whereinsaid detecting is by means of an immunoassay.
 16. The method of claim15, wherein said immunoassay is a radioimmunoassay, enzyme-linkedimmunosorbent assay (ELISA), flourescent immunoassay, or fluorescencepolarization immunoassay (FPIA).
 17. The method according to claim 15wherein said Shigella bacterium is Shigella flexneri.
 18. The methodaccording to claim 17 wherein said Shigella flexneri is strain 2457T andthe combination of conditions comprises:1) about 0.1% bile salt which isdeoxycholate or about 0.8% bile; 2) the temperature is about 37° C.; 3)in air; andthe culture is in early log phase.
 19. A diagnosticimmunoassay kit for detecting production of antibodies to Shigellabacteria or for detecting Shigella bacteria, comprising:a) a Shigellabacterium or a fragment thereof or antibodies thereto, wherein saidShigella bacterium has an enhanced antigenic property, and is harvestedfrom a culture of a Shigella species grown in vitro under a combinationof conditions comprising:1) about 0.05% to about 3% bile or about 0.025%to about 0.6% of one or more bile acids or salts thereof; 2) at atemperature between about 30° C. and about 42° C.; 3) in air or a gasmixture, wherein the gas mixture comprises i) about 5% to about 20% CO₂with about 80% to about 95% air; ii) about 5% to about 20% CO₂ withabout 80% to about 95% N₂ ; or iii) about 5% to about 10% O₂ with about10% to about 20% CO₂ with about 70% to about 85% N₂ ; and 4) a divalentcation chelator selected from the group consisting of 0 to about 100 μMof BAPTA/AM, 0 to about 10 mM of EGTA, and 0 about 100 μM of EGTA/AM,and b) at least one kit component selected from the group consisting ofmicrotiter plates, buffers to maintain pH and a conjugated antibody. 20.The diagnostic immunoassay kit according to claim 19, wherein said acidor salt thereof comprises deoxycholic acid or deoxycholate.
 21. Thediagnostic immunoassay kit of claim 19, wherein said Shigella bacteriumis a Shigella species selected from the group consisting of flexneri,sonnei, dysentariae and boydii.
 22. The diagnostic immunoassay kitaccording to claim 21, wherein said Shigella bacterium is Shigellaflexneri.
 23. A diagnostic immunoassay kit for detecting production ofantibodies to Shigella bacteria or for detecting Shigella bacteria,comprising:a) a Shigella bacterium or a fragment thereof or antibodiesthereto, wherein said Shigella bacterium has an enhanced antigenicproperty, and is harvested from a culture of a Shigella species grown invitro under a set of conditions comprising:1) a divalent cation chelatorselected from the group consisting of about 1.0 to about 25 μM ofBAPTA/AM, about 0.5 to about 10 mM of EGTA, and about 1 to about 100 μMof EGTA/AM; 2) at a temperature between about 30° and about 42° C.; and3) in air or an microaerophilic condition, wherein the microaerophiliccondition comprises i) about 5% to about 20% CO₂ with about 80% to about95% air; ii) about 5% to about 20% CO₂ with about 80% to about 95% N₂ ;or iii) about 5% to about 10% O₂ with about 10% to about 20% CO₂ withabout 70% to about 85% N₂, and b) at least one kit component selectedform the group consisting of microtiter plates, buffers to maintain pHand a conjugated antibody.
 24. The diagnostic immunoassay kit accordingto claim 23 wherein said Shigella bacterium is Shigella flexneri. 25.The diagnostic immunoassay kit according to claim 24 wherein saidShigella flexneri is strain 2457T and the combination of conditionscomprises:1) about 0.1% bile salt which is deoxycholate or about 0.3%bile; 2) the temperature is about 37° C.; 3) in air; andthe culture isin early log phase.